WO2014191590A1 - Double-inclusion method for the study of the histology and/or localisation of molecular markers in pluricellular specimens of microscopic dimensions - Google Patents

Double-inclusion method for the study of the histology and/or localisation of molecular markers in pluricellular specimens of microscopic dimensions Download PDF

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WO2014191590A1
WO2014191590A1 PCT/ES2014/000089 ES2014000089W WO2014191590A1 WO 2014191590 A1 WO2014191590 A1 WO 2014191590A1 ES 2014000089 W ES2014000089 W ES 2014000089W WO 2014191590 A1 WO2014191590 A1 WO 2014191590A1
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sample
minutes
inclusion
ethanol
specimens
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PCT/ES2014/000089
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Spanish (es)
French (fr)
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Rosa Ana PICAZO GONZÁLEZ
María de los Angeles SANCHEZ PEREZ
Belén SANCHEZ MALDONADO
María Teresa ENCINAS CEREZO
Juan Antonio GILABERT SANTOS
José María ROS RODRIGUEZ
Carmen Belén MARTÍNEZ MADRID
María del Pilar GARCIA PALENCIA
Pedro ARANDA ESPINOSA
Paola Stefanía TINETTI PINTO
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Universidad Complutense De Madrid
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures

Definitions

  • the present invention falls within the field of histotechnology. More specifically, it refers to a double histological inclusion procedure, composed of a biocompatible organic matrix preaggregation system and a paraffin inclusion system, which allows serial histological analyzes to be performed, and the location of molecular markers, particularly by immunohistochemistry, in specimens multicellular microscopic dimensions.
  • Double inclusion techniques of small specimens for subsequent structural or ultrastructural analysis have been known for approximately three decades.
  • the procedures commonly used include: fixation of the sample for times between twelve and twenty-four hours, followed by centrifugation in the case of cells, and preinclusion in agar, agarose, gelatin or bovine serum albumin (BSA), before or after centrifugation
  • BSA bovine serum albumin
  • the procedures end with dehydration in increasing concentrations of ethanol over time periods of one, or more hours (Harris MJ (1974). Cytotechnol. Bull. 1 1: 6-7; Moskaluk CA, Stoler MH (2002). Diag. Mol. Pathol. 1 1: 234-23).
  • Micron (38) 714-721 which comprise the fixation of the cells in suspension for one hour, the inclusion in agar, their centrifugation and post-fixation during one to two hours Subsequently, in the article by Taupin (Taupin, P (2008). Annals of Microscopy 8: 19-21), a procedure for processing suspension cells for electron microscopy is described. According to this protocol, the cells are fixed in glutaraldehyde for one hour at room temperature, are included in 5% gelatin or in 5% bovine serum albumin and subsequently crosslinked with fixing agents (paraformaldehyde and glutaraldehyde) for 1-1, 5 hours at room temperature.
  • fixing agents paraformaldehyde and glutaraldehyde
  • the pieces of gelatin or BSA are dehydrated in ethanol solutions at increasing concentration (50%, 75%, 95% and 100%, for 10, 20, 20 and 20 minutes, respectively) and finally included in araldite. This last procedure aims to avoid centrifugation of the sample, obtain a solid piece for study, keep the cells in suspension and establish a fixation time of the sample much shorter than usual.
  • the current state of the art includes centrifuging the cell sample to obtain a pellet, exposing it to the fixation solution for at least one hour, preinclusion in an aggregation matrix that facilitates the inclusion process and macroscopic sample manipulation, histological section and subsequent analysis.
  • the centrifugation processes of the sample and the fixation conditions are critical aspects of the methodology, which exert a significant influence on the maintenance of the cytostructure and the antigenic characteristics of the cells, essential for histological analysis and the location of molecular markers. , especially immunohistochemical, thereof.
  • the present invention relates to a double inclusion procedure for the histological and / or localization study of molecular markers of multicellular specimens of microscopic dimensions, which allows to maintain the three-dimensional spatial arrangement of the samples.
  • studies of molecular marker localization especially the immunohistochemical analyzes stand out.
  • specimen means the biological entity that is adopted as a sample, as well as the aggregate of cells of the same or different types, which may come from cell cultures, bacterial cultures, cell aspirates, among others , which is considered as a sample.
  • molecular marker means any biomolecule that can be associated or correlated with a genetic trait.
  • Biomolecules that can be molecular markers are peptides and proteins (for example, antigens and soenzymes) and nucleic acids (for example, known genes or fragments of unknown sequence and / or function).
  • immunohistochemistry refers to methods that are based on the use of mono or polyclonal antibodies, previously labeled by a chemical bond with an enzyme, that do not affect the ability of the antibody. to form a complex with an antigen, with utility in the detection of cellular antigens.
  • the process of the invention includes the pre-aggregation of multicellular samples of microscopic dimensions in agar matrix, optimized for paraffin inclusion and serial histological sections.
  • preinclusion in agar of cells from cell cultures is performed after obtaining a pellet by centrifugation, which causes the inconvenience of altering the original arrangement of the cells of the culture.
  • the centrifugation step has been eliminated and the inclusion in agar is carried out directly, which allows to maintain the original structure of the sample, for later morphological evaluation in the histological sections.
  • Another aspect of the invention relates to the period of time during which the fixation of the sample is carried out, which has been reduced to between 10 and 15 minutes, preferably being 15 minutes. It also refers to the temperature at which the fixation is performed, since better results have been obtained at temperatures between 2 and 8 ° C.
  • the invention includes shorter dehydration periods of the sample than are usually used according to the state of the art, since the process described herein includes 10 or 15 minute dehydration periods, preferably 10 minutes. , with each of the ethanol concentrations that are used which, in a preferred embodiment, are 3 concentrations in increasing order. Also, dehydration is preferably carried out at a temperature between 2 and 8 ° C.
  • the method comprises the following steps:
  • the influence of the conditions of the procedure on the maintenance of the structure of the specimens was examined by histological analysis and their antigenic integrity, since one of the applications of the procedure includes the subsequent realization of protein immunolocation techniques.
  • the procedure is suitable for the subsequent performance of numerous immunohistochemical techniques, such as immunolocation of protein antigens, or nucleic acids, among other molecular markers. Some of these techniques involve the incorporation of previous methods of antigen unmasking, which have been developed without difficulties, with an adequate final performance in immunolocation procedures.
  • this procedure can be used for the simultaneous processing and analysis of multiple molecular markers (both peptides and nucleic acids) in specimens of dimensions between 70 and 300 ⁇ and origin diverse.
  • molecular markers both peptides and nucleic acids
  • the process of the invention allows to provide samples of microscopic dimensions with a size that facilitates their macroscopic visualization and manipulation, by means of preinclusion or pre-aggregation thereof in a biocompatible matrix, which resists the paraffin inclusion process without alterations. .
  • experimental samples can be placed in the same plane within the inclusion block, so that they appear as a group in the subsequent histological sections.
  • tissue sample is placed and oriented in the desired position and the agar is deposited on it in a liquid (hot) state, allowing its subsequent solidification at room temperature.
  • tissue microarrays allow histological, immunohistochemical and in situ hybridization techniques to detect markers in a large number of samples from different tissues at one time.
  • the process of the invention provides other advantages: * It allows the histological and molecular markers (particularly the immunohistochemical) analysis of three-dimensional multicellular structures of microscopic dimensions, while maintaining maximum cytostructure and cellular antigenicity.
  • the described procedure is capable of being integrated, including the methodology and the solutions and reagents, in a kit that includes the elements necessary for its use in the processing of multicellular specimens of microscopic dimensions by double inclusion.
  • the kit may include buffers, enzymes, agents to prevent contamination, etc., as well as antibodies, peptides, nucleic acids or other molecules and reagents for molecular marker analysis or immunohistochemical analysis.
  • the kit can also include all the supports and containers necessary for carrying out the process of the invention.
  • microfotogs are presented corresponding to histological sections of cell aggregates, processed according to the procedure described and subjected to the fixation process, for 15 (A, B), 20 (C) and 30 minutes ( D), subsequently stained with hematoxylin and eosin.
  • FIG 3 images are shown showing the appearance of the mini agar blocks, which contain numerous groups of cell aggregates, after the process of inclusion in paraffin (A) and in the complete block, prepared for microtome cutting (B).
  • the cuts of 3 ⁇ , are arranged on slides for histological or immunohistochemical analysis (C).
  • C histological or immunohistochemical analysis
  • D Staining with hematoxylin-eosin, allowed to evaluate the cytological characteristics of multicellular structures, during the completion of the complete procedure (E).
  • the primary antibodies used in the immunolocation of these antigens were anti-K ⁇ 67 (Leica Biosystems, Ref. KI67-MM1 -L-CE) and anti-caspase 3-active (R&D, ref. AF835). Images obtained with the optical microscope (Olympus BX40 Miroscope), with an image analysis program.
  • Example 1 Structural analysis of cellular aggregates generated from the ovarian cell culture of the ovine species.
  • a culture of ovarian tissue cells of the ovine species was used.
  • the cells were obtained from prepubertal lamb ovaries, by an enzymatic isolation procedure with collagenase, followed by mechanical disintegration. From the resulting cell suspension, 500,000 living cells were grown per 1.9 cm 2 of growth surface, in plates of 24-well culture (Nunclon Delta Surface, Nunc, Thermo Fisher Scientific ref. 142475), with M199 medium (Sigma Aldrich Chemistry ref. M7528), supplemented with antibiotic and antifungal, bovine serum albumin, insulin, transferrin and selenium, at 37 ° C, 5% CO2 and humidity at saturation, for 4 weeks. During this period, a weekly sampling point was made for histological analysis. Cellular aggregates reached, on average, diameters of 70-120 ⁇ in the first week of culture, 120-200 ⁇ in the second week, and 200-300 ⁇ in the third and fourth weeks of culture.
  • phosphate buffer solution 500 ⁇ of DPBS Sigma Aldrich Qu ⁇ mica, ref. D8537
  • the fixing system comprised the following steps. 500 ⁇ of 4% paraformaldehyde solution (Sigma Aldrich Chemistry, ref. P6148) was dispensed in PBS (DPBS Sigma Aldrich Chemistry, ref. D8537) at pH 7.4, in each culture well. The samples were divided into 2 groups, so that half of them were kept for 10 minutes and the other half for 15 minutes in paraformaldehyde at a temperature of 2-8 ° C. At the end of this period, the fixation solution was removed and a 5 minute double wash was performed, with phosphate buffer solution (DPBS) at 2-8 ° C. 3. - Dehydration in increasing concentration of ethanol.
  • step 1 In half of the samples, from each culture well, the phosphate buffer solution used in step 1 was removed and, at intervals of 10 minutes, 500 ⁇ of ethanol (Merck Millipore Absolute) were successively dispensed and replaced. , ref. 1009832500) at 30%, 50% and 70%, in ultra pure water (MilliQ). The entire dehydration process was carried out at 2-8 ° C. 3.2.- The other half of the samples were dehydrated following the pattern described in section 3.1., But keeping the samples for 15 minutes at each concentration of ethanol.
  • ethanol Merck Millipore Absolute
  • the preinclusion process included the steps described below:
  • agar solution for bacteriological culture (Oxoid Ltd. Hampshire, England, ref. LP001), 1.5% in distilled water, was prepared. The mixture was kept under stirring on a thermal plate until it reached 85 ° C, approximate melting temperature of the agar, avoiding boiling. Its use began when the solution was completely transparent. The solution was kept in a state of gentle and permanent agitation during the time in which the preinclusion process took place, at a temperature between 82 and 100 ° C.
  • a polypropylene mold 5 mm in diameter and 3 mm deep was prepared as a plastic support.
  • the mold base was completely covered with a volume of 40 ⁇ 40 agar solution, preventing the formation of bubbles.
  • Samples were transferred from 70% ethanol solution, to the agar base, coinciding with the beginning of gelation thereof by thermal descent ( Figure 2C, D), using capillary micropipettes adapted to a syringe. Then, under stereoscopic microscope, the excess ethanol was removed from the samples, with capillary micropipettes adapted to a syringe and with Whatmann absorbent paper threads.
  • Example 2 Immunolocalization of the marker antigen of Ki67 proliferating cells in pseudo-follicular structures derived in vitro from the co-culture of ovarian primordial follicles with stroma-interstitial cells.
  • ovarian pseudo-follicles in development in vitro, which were maintained in culture in a defined medium, at 37 ° C, with 5% CO 2 and humidity at saturation. These structures adopted an approximately spherical three-dimensional morphology, with diameters between 70 and 280 ⁇ throughout the cultivation period.
  • Sample preparation and fixation, dehydration, agar matrix pre-aggregation, paraffin inclusion, block and cut preparation procedures were carried out as detailed. in section 5 of Example 1, using 15-minute sample fixation times and 10-minute dehydration periods with each ethanol concentration. On histological sections of 3 ⁇ , immunolocation of the Ki67 proliferating cell antigen was carried out.
  • a monoclonal antibody (Anti-Ki67, Leica Biosystems, Ref. KI67-MM1-L-CE) was used following the immunohistochemical analysis procedure detailed below.
  • a technique validated by researchers of this group was used in the Histology and Pathology Laboratory of the Veterinary Clinical Hospital of the Complutense University of Madrid, using a commercial kit (Novolink Polymer Detection System ® Novocastra, Leica Microsystems, Barcelona Ref .: RE -7140-K), which includes the following reagents:
  • TBS tris buffer solution
  • Example 3 Immunolocalization of the enzyme caspase 3- activa, marker of cells that have started the apoptosis process, in pseudo-follicular structures derived in vitro from co-culture of ovarian primordial follicles with stroma-interstitial cells.

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Abstract

The invention relates to a method involving the double inclusion of pluricellular specimens of microscopic dimensions (70-300 μιη), which allows the three-dimensional spatial arrangement of the samples to be maintained in order to subsequently carry out histological, immunohistochemical and/or localisation studies on molecular markers. The method includes preparing the sample by means of washes with a buffer solution; fixing the sample for 10-15 minutes at a temperature of between 2 and 8°C; dehydrating the sample with increasing concentrations of ethanol for 10-15 minutes respectively, at a temperature of between 2 and 8°C; and pre-inclusion in agar and inclusion in paraffin, without centrifuging the sample in any of the steps of the method. The invention also includes a kit with the elements necessary for the implementation of the method described in the invention.

Description

TÍTULO  TITLE
Procedimiento de doble inclusión para el estudio histológico y/o de localización de marcadores moleculares en especímenes pluricelulares de dimensiones microscópicas  Double inclusion procedure for histological study and / or location of molecular markers in multicellular specimens of microscopic dimensions
SECTOR DE LA TÉCNICA SECTOR OF THE TECHNIQUE
La presente invención se encuadra en el sector de la histotecnología. Más concretamente se refiere a un procedimiento de doble inclusión histológica, compuesto por un sistema de preagregación en matriz orgánica biocompatible y de inclusión en parafina, que permite la realización de análisis histológicos seriados, y de localización de marcadores moleculares, particularmente mediante inmunohistoquímica, en especímenes pluricelulares de dimensiones microscópicas. The present invention falls within the field of histotechnology. More specifically, it refers to a double histological inclusion procedure, composed of a biocompatible organic matrix preaggregation system and a paraffin inclusion system, which allows serial histological analyzes to be performed, and the location of molecular markers, particularly by immunohistochemistry, in specimens multicellular microscopic dimensions.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
Las técnicas de doble inclusión de especímenes de pequeñas dimensiones para el análisis estructural o ultraestructural posterior son conocidas desde hace aproximadamente tres décadas. Los procedimientos utilizados habitualmente incluyen: la fijación de la muestra durante tiempos comprendidos entre doce y veinticuatro horas, seguida de centrifugación si se trata de células, y la preinclusión en agar, agarosa, gelatina o albúmina sérica bovina (BSA), antes o después de la centrifugación. Los procedimientos finalizan con la deshidratación en concentraciones crecientes de etanol en periodos de tiempo de una, o más horas (Harris M.J. (1974). Cytotechnol. Bull. 1 1 :6-7; Moskaluk C.A., Stoler M.H. (2002). Diag. Mol. Pathol. 1 1 : 234- 23). Double inclusion techniques of small specimens for subsequent structural or ultrastructural analysis have been known for approximately three decades. The procedures commonly used include: fixation of the sample for times between twelve and twenty-four hours, followed by centrifugation in the case of cells, and preinclusion in agar, agarose, gelatin or bovine serum albumin (BSA), before or after centrifugation The procedures end with dehydration in increasing concentrations of ethanol over time periods of one, or more hours (Harris MJ (1974). Cytotechnol. Bull. 1 1: 6-7; Moskaluk CA, Stoler MH (2002). Diag. Mol. Pathol. 1 1: 234-23).
En fechas recientes, se han desarrollado metodologías dirigidas al análisis ultraestructural de especímenes pluricelulares por microscopía electrónica de transmisión, en las que se reduce a una hora el tiempo de exposición de las muestras a las soluciones de fijación y se realiza una doble inclusión utilizando diferentes soportes. Como exponentes de esta aproximación, destacan los procedimientos descritos por diversos autores (Dvorak A.M., y cois. (1991). American Journal of Pathology, 138 (1): 69-82; Meló R.C.N., y cois., (2005). Trafíic 6: 1047-1057; Meló R.C.N., y cois., (2007). Micron (38) 714-721), que comprenden la fijación de las células en suspensión durante una hora, la inclusión en agar, su centrifugación y la postfijación durante una a dos horas. Posteriormente, en el artículo de Taupin (Taupin, P (2008). Annals of Microscopy 8: 19-21), se describe un procedimiento para procesar células en suspensión para microscopía electrónica. Según este protocolo, las células se fijan en glutaraldehído durante una hora a temperatura ambiente, se incluyen en gelatina al 5% o en albúmina sérica bovina al 5% y posteriormente se entrecruzan con agentes fijadores (paraformaldehído y glutaraldehído) durante 1-1 ,5 horas a temperatura ambiente. Las piezas de gelatina o BSA se deshidratan en soluciones de etanol a concentración creciente (50%, 75%. 95% y 100%, durante 10, 20, 20 y 20 minutos, respectivamente) y finalmente se incluyen en araldita. Este último procedimiento pretende evitar la centrifugación de la muestra, obtener una pieza sólida para su estudio, mantener las células en suspensión y establecer un tiempo de fijación de la muestra mucho más corto de lo habitual. Recently, methodologies aimed at the ultrastructural analysis of multicellular specimens by transmission electron microscopy have been developed, in which the exposure time of the samples to fixation solutions and double inclusion is performed using different supports. As exponents of this approach, the procedures described by various authors stand out (Dvorak AM, et al. (1991). American Journal of Pathology, 138 (1): 69-82; Meló RCN, et al., (2005). Trafíic 6: 1047-1057; Meló RCN, et al., (2007). Micron (38) 714-721), which comprise the fixation of the cells in suspension for one hour, the inclusion in agar, their centrifugation and post-fixation during one to two hours Subsequently, in the article by Taupin (Taupin, P (2008). Annals of Microscopy 8: 19-21), a procedure for processing suspension cells for electron microscopy is described. According to this protocol, the cells are fixed in glutaraldehyde for one hour at room temperature, are included in 5% gelatin or in 5% bovine serum albumin and subsequently crosslinked with fixing agents (paraformaldehyde and glutaraldehyde) for 1-1, 5 hours at room temperature. The pieces of gelatin or BSA are dehydrated in ethanol solutions at increasing concentration (50%, 75%, 95% and 100%, for 10, 20, 20 and 20 minutes, respectively) and finally included in araldite. This last procedure aims to avoid centrifugation of the sample, obtain a solid piece for study, keep the cells in suspension and establish a fixation time of the sample much shorter than usual.
En relación con la aplicación de métodos de doble inclusión al análisis estructural e inmunohistoquímico de especímenes de dimensiones microscópicas, éstos se han dirigido mayoritariamente a la investigación con células en cultivo. Entre los procedimientos, destaca el publicado por Gruber y cois. (Gruber HE, y cois., (2009). Biotechnic & Histochemistry 84 (6): 283- 286), en el que se especifica que, cuando la muestra contiene pocos estratos celulares, se requieren periodos reducidos de exposición a la solución de fijación, de una hora, previa centrifugación de la muestra. Estos mismos autores, citan otros procedimientos que también implican un hora de fijación (Tapp, H. y cois., (2008) Arthritis Research & Therapy (W) 4 - R89). En resumen, el estado actual de la técnica incluye la centrifugación de la muestra celular para obtener un pellet, la exposición del mismo a la solución de fijación durante al menos una hora, la preinclusión en una matriz de agregación que facilite el proceso de inclusión y manipulación macroscópica de la muestra, el corte histológico y el análisis posterior. Los procesos de centrifugación de la muestra y las condiciones de fijación son aspectos críticos de la metodología, que ejercen una influencia notable sobre el mantenimiento de la citoestructura y de las características antigénicas de las células, esenciales para el análisis histológico y la localización de marcadores moleculares, especialmente inmunohistoquímico, de las mismas. Regarding the application of double inclusion methods to the structural and immunohistochemical analysis of specimens of microscopic dimensions, these have mostly been directed to research with cells in culture. Among the procedures, the one published by Gruber and cois stands out. (Gruber HE, et al., (2009). Biotechnic & Histochemistry 84 (6): 283-286), in which it is specified that, when the sample contains few cell strata, reduced periods of exposure to the solution are required. fixation, one hour, after centrifugation of the sample. These same authors cite other procedures that also involve an hour of fixation (Tapp, H. et al., (2008) Arthritis Research & Therapy (W) 4 - R89). In summary, the current state of the art includes centrifuging the cell sample to obtain a pellet, exposing it to the fixation solution for at least one hour, preinclusion in an aggregation matrix that facilitates the inclusion process and macroscopic sample manipulation, histological section and subsequent analysis. The centrifugation processes of the sample and the fixation conditions are critical aspects of the methodology, which exert a significant influence on the maintenance of the cytostructure and the antigenic characteristics of the cells, essential for histological analysis and the location of molecular markers. , especially immunohistochemical, thereof.
EXPLICACIÓN DE LA INVENCIÓN EXPLANATION OF THE INVENTION
La presente invención se refiere a un procedimiento de doble inclusión para el estudio histológico y/o de localización de marcadores moleculares de especímenes pluricelulares de dimensiones microscópicas, que permite mantener la disposición espacial tridimensional de las muestras. Entre los estudios de localización de marcadores moleculares, destacan especialmente los análisis inmunohistoquímicos. The present invention relates to a double inclusion procedure for the histological and / or localization study of molecular markers of multicellular specimens of microscopic dimensions, which allows to maintain the three-dimensional spatial arrangement of the samples. Among the studies of molecular marker localization, especially the immunohistochemical analyzes stand out.
En la presente memoria descriptiva, se entiende por "espécimen" la entidad biológica que se adopta como muestra, así como el agregado de células del mismo tipo o de tipos diferentes, que puede proceder de cultivos celulares, cultivos bacterianos, aspirados celulares, entre otros, que se considera como muestra. In the present specification, "specimen" means the biological entity that is adopted as a sample, as well as the aggregate of cells of the same or different types, which may come from cell cultures, bacterial cultures, cell aspirates, among others , which is considered as a sample.
Asimismo, se entiende por "marcador molecular" toda biomolécula que se puede asociar o correlacionar con un rasgo genético. Las biomoléculas que pueden ser marcadores moleculares son los péptidos y las proteínas (por ejemplo, antígenos e ¡soenzimas) y los ácidos nucleicos (por ejemplo, genes conocidos o fragmentos de secuencia y/o función desconocida). El término "inmunohistoquímica", tal y como se utiliza en esta memoria, se refiere a los métodos que se basan en la utilización de anticuerpos mono o policlonales, previamente marcados mediante un enlace químico con una enzima, que no afectan a la capacidad del anticuerpo para formar un complejo con un antígeno, con utilidad en la detección de antígenos celulares. Likewise, "molecular marker" means any biomolecule that can be associated or correlated with a genetic trait. Biomolecules that can be molecular markers are peptides and proteins (for example, antigens and soenzymes) and nucleic acids (for example, known genes or fragments of unknown sequence and / or function). The term "immunohistochemistry," as used herein, refers to methods that are based on the use of mono or polyclonal antibodies, previously labeled by a chemical bond with an enzyme, that do not affect the ability of the antibody. to form a complex with an antigen, with utility in the detection of cellular antigens.
El procedimiento de la invención incluye la preagregación de muestras pluricelulares de dimensiones microscópicas en matriz de agar, optimizado para la inclusión en parafina y los cortes histológicos seriados. En los laboratorios de histotecnología, la preinclusión en agar de células procedentes de cultivos celulares se realiza después de la obtención de un pellet por centrifugación, lo que acarrea el inconveniente de alterar la disposición original de las células del cultivo. En el procedimiento de la invención se ha eliminado el paso de centrifugación y se realiza directamente la inclusión en agar, lo que permite mantener la estructura original de la muestra, para su valoración morfológica posterior en las secciones histológicas. The process of the invention includes the pre-aggregation of multicellular samples of microscopic dimensions in agar matrix, optimized for paraffin inclusion and serial histological sections. In histotechnology laboratories, preinclusion in agar of cells from cell cultures is performed after obtaining a pellet by centrifugation, which causes the inconvenience of altering the original arrangement of the cells of the culture. In the process of the invention the centrifugation step has been eliminated and the inclusion in agar is carried out directly, which allows to maintain the original structure of the sample, for later morphological evaluation in the histological sections.
Otro aspecto de la invención se refiere al periodo de tiempo durante el que se realiza la fijación de la muestra, que se ha reducido a entre 10 y 15 minutos, siendo de preferencia de 15 minutos. También se refiere a la temperatura a la que se realiza la fijación, puesto que se han obtenido mejores resultados a temperaturas entre 2 y 8°C. Por otro lado, la invención incluye periodos de deshidratación de la muestra más reducidos de los que se utilizan habitualmente según el estado de la técnica, dado que el procedimiento que aquí se describe incluye periodos de deshidratación de 10 o 15 minutos, preferentemente de 10 minutos, con cada una de las concentraciones de etanol que se utilizan que, en una realización preferida, son 3 concentraciones en orden creciente. Así mismo, la deshidratación se realiza preferentemente a una temperatura de entre 2 y 8°C. En una realización preferida, el procedimiento comprende los siguientes pasos: Another aspect of the invention relates to the period of time during which the fixation of the sample is carried out, which has been reduced to between 10 and 15 minutes, preferably being 15 minutes. It also refers to the temperature at which the fixation is performed, since better results have been obtained at temperatures between 2 and 8 ° C. On the other hand, the invention includes shorter dehydration periods of the sample than are usually used according to the state of the art, since the process described herein includes 10 or 15 minute dehydration periods, preferably 10 minutes. , with each of the ethanol concentrations that are used which, in a preferred embodiment, are 3 concentrations in increasing order. Also, dehydration is preferably carried out at a temperature between 2 and 8 ° C. In a preferred embodiment, the method comprises the following steps:
a) preparación de la muestra mediante lavados con solución tamponada y sin centrifugación previa;  a) sample preparation by washing with buffered solution and without prior centrifugation;
b) fijación de la muestra con un agente fijador durante un periodo de tiempo comprendido entre 10 y 15 minutos, a temperatura de 2-8°C; c) deshidratación de la muestra con concentraciones crecientes de etanol durante 10-15 minutos cada una, a una temperatura de 2-8°C; d) preinclusión de la muestra en agar sin centrifugación, transfiriendo dicha muestra desde la solución de etanol de deshidratación a una base de agar preparada previamente y en estado de gelificación por descenso térmico;  b) fixation of the sample with a fixing agent for a period of time between 10 and 15 minutes, at a temperature of 2-8 ° C; c) dehydration of the sample with increasing concentrations of ethanol for 10-15 minutes each, at a temperature of 2-8 ° C; d) preinclusion of the sample in agar without centrifugation, transferring said sample from the dehydration ethanol solution to a previously prepared agar base and in gelation state by thermal descent;
e) inclusión en parafina.  e) inclusion in paraffin.
Por otra parte, en el proceso de validación, se examinó la influencia de las condiciones del procedimiento sobre el mantenimiento de la estructura de los especímenes mediante el análisis histológico y de la integridad antigénica de los mismos, puesto que una de las aplicaciones del procedimiento comprende la realización posterior de técnicas de inmunolocalización de proteínas. El procedimiento es adecuado para la realización posterior de numerosas técnicas de inmunohistoquímica, como son la inmunolocalización de antígenos proteicos, o de ácidos nucleicos, entre otros marcadores moleculares. Algunas de estas técnicas implican la incorporación de métodos previos de desenmascaramiento antigénico, que se han desarrollado sin dificultades, con un rendimiento final adecuado en los procedimientos de inmunolocalización. On the other hand, in the validation process, the influence of the conditions of the procedure on the maintenance of the structure of the specimens was examined by histological analysis and their antigenic integrity, since one of the applications of the procedure includes the subsequent realization of protein immunolocation techniques. The procedure is suitable for the subsequent performance of numerous immunohistochemical techniques, such as immunolocation of protein antigens, or nucleic acids, among other molecular markers. Some of these techniques involve the incorporation of previous methods of antigen unmasking, which have been developed without difficulties, with an adequate final performance in immunolocation procedures.
Además de las aplicaciones inmunohistoquímicas, este procedimiento puede ser utilizado para el procesamiento y análisis simultáneo de múltiples marcadores moleculares (tanto péptidos como ácidos nucleicos) en especímenes de dimensiones comprendidas entre 70 y 300 μιη y origen diverso. Como ejemplos, se pueden citar los procedentes de embriones en estadios preimplantacionales, cuerpos embrioides o neuroesferas, derivados de diferenciación de células pluripotentes, fases larvarias o adultas de parásitos, cultivos bacterianos, agregados de células procedentes de cultivos celulares o de aspirados celulares y/o modelos animales microscópicos, entre otros. In addition to immunohistochemical applications, this procedure can be used for the simultaneous processing and analysis of multiple molecular markers (both peptides and nucleic acids) in specimens of dimensions between 70 and 300 μιη and origin diverse. As examples, those from embryos in preimplantation stages, embryoid or neurosphere bodies, derivatives of differentiation of pluripotent cells, larval or adult phases of parasites, bacterial cultures, aggregates of cells from cell cultures or cell aspirates and / or microscopic animal models, among others.
El procedimiento de la invención, permite proporcionar a las muestras de dimensiones microscópicas un tamaño que facilita su visualización macroscópica y su manipulación, por medio de la preinclusión o preagregación de las mismas en una matriz biocompatible, que resiste sin alteraciones el proceso de inclusión en parafina. The process of the invention allows to provide samples of microscopic dimensions with a size that facilitates their macroscopic visualization and manipulation, by means of preinclusion or pre-aggregation thereof in a biocompatible matrix, which resists the paraffin inclusion process without alterations. .
En este sentido, se puede preagregar un número elevado de muestras en cada bloque de preinclusión y posterior inclusión, lo que permite el análisis estructural y de marcadores moleculares simultáneo en grupo (i.e. por grupo experimental o tratamiento). In this sense, a large number of samples can be pre-aggregated in each preinclusion and subsequent inclusion block, which allows simultaneous structural and molecular marker analysis in groups (i.e. by experimental group or treatment).
Además, las muestras experimentales pueden situarse en el mismo plano dentro del bloque de inclusión, con el fin de que aparezcan en grupo en los cortes histológicos posteriores. In addition, the experimental samples can be placed in the same plane within the inclusion block, so that they appear as a group in the subsequent histological sections.
La eliminación del proceso de centrifugación de la muestra, la reducción del tiempo de exposición a la solución de fijación y el llevar a cabo el procedimiento de fijación y deshidratación a temperatura de refrigeración, permiten obtener muestras con una adecuada citoestructura para el análisis histológico y que mantienen las características antigénicas que permiten la realización de estudios de inmunolocalización de marcadores múltiples, así como de otros análisis de marcadores moleculares. The elimination of the centrifugation process of the sample, the reduction of the exposure time to the fixation solution and the carrying out of the fixation and dehydration procedure at refrigeration temperature, allow to obtain samples with an adequate cyto-structure for histological analysis and that they maintain the antigenic characteristics that allow the realization of immunolocalization studies of multiple markers, as well as other analyzes of molecular markers.
Durante el desarrollo del procedimiento, se determinó que las exposiciones a la solución de fijación, durante periodos de tiempo iguales o superiores a 20 minutos, tenían como consecuencia la alteración de las características citológicas, con una elevada incidencia de crenación celular y retracción de las estructuras tridimensionales, con pérdida de uniones intercelulares(figura 1 C, D)y alteración en la antigenicidad celular, que se constató posteriormente al realizar técnicas de inmunolocalización de diversos marcadores de rutina. During the development of the procedure, it was determined that exposures to the fixation solution, for periods of time equal to or greater than 20 minutes, they had as a consequence the alteration of the cytological characteristics, with a high incidence of cell creation and retraction of the three-dimensional structures, with loss of intercellular junctions (Figure 1 C, D) and alteration in cellular antigenicity, which was subsequently verified at Perform immunolocation techniques of various routine markers.
Desde un punto de vista genérico, el procedimiento proporciona de forma consistente las ventajas y beneficios del empleo de las técnicas denominadas de "doble inclusión" en agar y parafina para el estudio histológico que se realizan ocasionalmente en los laboratorios de histología con varios fines: From a generic point of view, the procedure consistently provides the advantages and benefits of the use of so-called "double inclusion" techniques in agar and paraffin for histological study that are occasionally performed in histology laboratories for several purposes:
* Orientación de muestras de pequeñas dimensiones para facilitar la sección adecuada: en ocasiones las muestras quirúrgicas remitidas a los laboratorios de anatomía patológica para su diagnóstico son de pequeño tamaño, lo que dificulta la realización de una orientación adecuada de la misma para realizar las secciones. La muestra de tejido es colocada y orientada en la posición deseada y se deposita sobre ella el agar en estado líquido (caliente), permitiendo su solidificación posterior a temperatura ambiente. * Preparación de agregados de células procedentes de cultivos o de aspirados celulares para su procesado histológico rutinario. * Orientation of small samples to facilitate the appropriate section: sometimes the surgical samples sent to the pathology laboratories for diagnosis are small, making it difficult to perform an appropriate orientation to perform the sections. The tissue sample is placed and oriented in the desired position and the agar is deposited on it in a liquid (hot) state, allowing its subsequent solidification at room temperature. * Preparation of cell aggregates from cultures or cell aspirates for routine histological processing.
* Preparación de microarrays a partir de líneas celulares: los microarrays de tejidos permiten realizar técnicas histológicas, inmunohistoquímicas y de hibridación in situ para la detección de marcadores en un número elevado de muestras procedentes de diferentes tejidos en una sola vez. * Preparation of microarrays from cell lines: tissue microarrays allow histological, immunohistochemical and in situ hybridization techniques to detect markers in a large number of samples from different tissues at one time.
* Posibilidad de predeterminar la ubicación exacta de un espécimen microscópico y de proporcionar coordenadas de emplazamiento exacto, para los protocolos de microscopía motorizada bidimensional. * Possibility of predetermining the exact location of a microscopic specimen and providing exact location coordinates, for two-dimensional motorized microscopy protocols.
Además, el procedimiento de la invención aporta otras ventajas: * Permite el análisis histológico y de marcadores moleculares (particularmente el inmunohistoquímico), de estructuras multicelulares tridimensionales de dimensiones microscópicas, manteniendo al máximo la citoestructura y la antigenicidad celular. In addition, the process of the invention provides other advantages: * It allows the histological and molecular markers (particularly the immunohistochemical) analysis of three-dimensional multicellular structures of microscopic dimensions, while maintaining maximum cytostructure and cellular antigenicity.
* Permite el análisis histológico y de marcadores moleculares (especialmente el inmunohistoquímico), de un número elevado de muestras de forma simultánea. * Allows histological analysis and molecular markers (especially immunohistochemistry) of a large number of samples simultaneously.
* Permite realizar un screening antigénico múltiple, para cada espécimen o grupo de muestras. Esto representa ventajas aplicativas directas respecto a las técnicas de inmunofluorescencia confocal, en las que pueden localizarse simultáneamente un número limitado de antígenos. * Allows multiple antigen screening, for each specimen or group of samples. This represents direct application advantages over confocal immunofluorescence techniques, in which a limited number of antigens can be located simultaneously.
* Permite realizar screening de expresión génica múltiple, mediante hibridación in situ sobre cortes histológicos de muestras multicelulares fijadas e incluidas en parafina, según procedimientos de localización ya establecidos en el estado de la técnica. * It allows screening of multiple gene expression, by in situ hybridization on histological sections of multicellular samples fixed and included in paraffin, according to localization procedures already established in the state of the art.
El procedimiento descrito es susceptible de ser integrado, incluyendo la metodología y las soluciones y reactivos, en un kit que comprenda los elementos necesarios para su uso en el procesamiento de especímenes pluricelulares de dimensiones microscópicas mediante doble inclusión. El kit puede incluir tampones, enzimas, agentes para prevenir la contaminación, etc., así como anticuerpos, péptidos, ácidos nucleicos u otras moléculas y reactivos para el análisis de marcadores moleculares o el análisis inmunohistoquímico. El kit también puede incluir todos los soportes y recipientes necesarios para la realización del procedimiento de la invención. The described procedure is capable of being integrated, including the methodology and the solutions and reagents, in a kit that includes the elements necessary for its use in the processing of multicellular specimens of microscopic dimensions by double inclusion. The kit may include buffers, enzymes, agents to prevent contamination, etc., as well as antibodies, peptides, nucleic acids or other molecules and reagents for molecular marker analysis or immunohistochemical analysis. The kit can also include all the supports and containers necessary for carrying out the process of the invention.
Los campos de conocimiento en los que el procedimiento tiene gran interés son: la biología celular, la parasitología, la embriología en diversas especies, la acuicultura, la investigación sobre impacto medioambiental de contaminantes químicos, el diagnóstico anatomopatológico cuando las biopsias son de reducidas dimensiones, la reproducción asistida humana, la investigación sobre patologías de base genética y su diagnóstico en embriones preimplantacionales, los sistemas de inducción y diferenciación de células pluripotentes, el análisis de marcadores múltiples en modelos animales de dimensiones reducidas o microscópicas, las acciones de fármacos, agentes tóxicos y contaminantes medioambientales en la expresión de proteínas en organismos pluricelulares microscópicos, entre muchas otras. The fields of knowledge in which the procedure is of great interest are: cell biology, parasitology, embryology in various species, aquaculture, research on the environmental impact of chemical pollutants, pathological diagnosis when biopsies are small, human assisted reproduction, research on genetically based pathologies and their diagnosis in preimplantation embryos, induction systems and cell differentiation pluripotent, the analysis of multiple markers in animal models of reduced or microscopic dimensions, the actions of drugs, toxic agents and environmental pollutants in the expression of proteins in microscopic multicellular organisms, among many others.
DESCRIPCIÓN DE LOS DIBUJOS DESCRIPTION OF THE DRAWINGS
I. En la figura 1 , se presentan microfotog rafias correspondientes a cortes histológicos, de agregados celulares, procesados de acuerdo con el procedimiento descrito y sometidos al proceso de fijación, durante 15 (A, B), 20 (C) y 30 minutos (D), posteriormente teñidos con hematoxilina y eosina. I. In Figure 1, microfotogs are presented corresponding to histological sections of cell aggregates, processed according to the procedure described and subjected to the fixation process, for 15 (A, B), 20 (C) and 30 minutes ( D), subsequently stained with hematoxylin and eosin.
II. En la figura 2, se presentan imágenes correspondientes a las etapas del procedimiento, para el procesado de agregados de células ováricas de la especie ovina (120-200 μηη). Los procedimientos de lavado, fijación y deshidratación de los agregados celulares (A, B) así como la preagregación en matriz de agar (C, D), se llevaron a cabo bajo microscopio estereoscópico (Microscopio estereoscópico Nikon SMZ800, 10X6.3). II. In figure 2, images corresponding to the steps of the procedure are presented, for the processing of ovarian cell aggregates of the ovine species (120-200 μηη). The procedures for washing, fixing and dehydration of cell aggregates (A, B) as well as pre-aggregation in agar matrix (C, D), were carried out under stereoscopic microscope (Nikon SMZ800 Stereoscopic Microscope, 10X6.3).
III. En la figura 3, se presentan imágenes que muestran la apariencia de los mini-bloques de agar, que contienen grupos numerosos de agregados celulares, después del proceso de inclusión en parafina (A) y en el bloque completo, preparados para el corte en microtomo (B). Los cortes de 3 μιτι, se dispusieron sobre portaobjetos para el análisis histológico o inmunohistoquímico (C). El procedimiento permite la inmunolocalización de un elevado número de marcadores, de forma simultánea en tantos especímenes, como sea necesario (D). La tinción con hematoxilina-eosina, permitió evaluar las características citológicas de las estructuras pluricelulares, durante la puesta a punto del procedimiento completo (E). III. In figure 3, images are shown showing the appearance of the mini agar blocks, which contain numerous groups of cell aggregates, after the process of inclusion in paraffin (A) and in the complete block, prepared for microtome cutting (B). The cuts of 3 μιτι, are arranged on slides for histological or immunohistochemical analysis (C). The procedure allows immunolocation of a high number of markers, simultaneously in as many specimens, as necessary (D). Staining with hematoxylin-eosin, allowed to evaluate the cytological characteristics of multicellular structures, during the completion of the complete procedure (E).
IV. En la figura 4, se presentan microfotografías, que muestran la inmunolocalización del antígeno de proliferación celular Ki67 (A-IV. In Figure 4, photomicrographs are presented, showing the immunolocation of the Ki67 cell proliferation antigen (A-
C) y del enzima caspasa 3-activa (D-F), implicada en el inicio del proceso de apoptosis celular. Los anticuerpos primarios utilizados en la inmunolocalización de estos antígenos fueron anti-K¡67 (Leica Biosystems, Ref. KI67-MM1 -L-CE) y anti caspasa 3-activa (R&D, ref. AF835). Imágenes obtenidas al microscopio óptico (Miroscopio Olympus BX40), con un programa de análisis de imagen. C) and the enzyme caspase 3-active (D-F), involved in the beginning of the process of cell apoptosis. The primary antibodies used in the immunolocation of these antigens were anti-K¡67 (Leica Biosystems, Ref. KI67-MM1 -L-CE) and anti-caspase 3-active (R&D, ref. AF835). Images obtained with the optical microscope (Olympus BX40 Miroscope), with an image analysis program.
MODO DE REALIZACIÓN MODE OF REALIZATION
Los siguientes ejemplos sirven para ilustrar la naturaleza de la presente invención y se incluyen únicamente con fines ilustrativos que no deben interpretarse como limitativos de la invención. The following examples serve to illustrate the nature of the present invention and are included for illustrative purposes only that should not be construed as limiting the invention.
Ejemplo 1.- Análisis estructural de agregados celulares generados a partir del cultivo de células ováricas de la especie ovina. Example 1.- Structural analysis of cellular aggregates generated from the ovarian cell culture of the ovine species.
Se utilizó un cultivo de células de tejido ovárico de la especie ovina. Las células se obtuvieron de ovarios de cordera prepúber, mediante un procedimiento de aislamiento enzimático con colagenasa, seguido de disgregación mecánica. De la suspensión celular resultante, se cultivaron 500.000 células vivas por 1.9 cm2 de superficie de crecimiento, en placas de cultivo de 24 pocilios (Nunclon Delta Surface, Nunc, Thermo Fisher Scientific ref. 142475), con medio M199 (Sigma Aldrich Química ref.M7528), suplementado con antibiótico y antimicótico, albúmina sérica bovina, insulina, transferrina y selenio, a 37°C, 5% de CO2 y humedad a saturación, durante 4 semanas. Durante este periodo, se efectuó un punto de muestreo semanal, para el análisis histológico. Los agregados celulares alcanzaban, en promedio, diámetros de 70-120 μιη en la primera semana de cultivo, 120-200 μιη en la segunda semana, y 200-300 μιη en la tercera y cuarta semanas de cultivo. A culture of ovarian tissue cells of the ovine species was used. The cells were obtained from prepubertal lamb ovaries, by an enzymatic isolation procedure with collagenase, followed by mechanical disintegration. From the resulting cell suspension, 500,000 living cells were grown per 1.9 cm 2 of growth surface, in plates of 24-well culture (Nunclon Delta Surface, Nunc, Thermo Fisher Scientific ref. 142475), with M199 medium (Sigma Aldrich Chemistry ref. M7528), supplemented with antibiotic and antifungal, bovine serum albumin, insulin, transferrin and selenium, at 37 ° C, 5% CO2 and humidity at saturation, for 4 weeks. During this period, a weekly sampling point was made for histological analysis. Cellular aggregates reached, on average, diameters of 70-120 μιη in the first week of culture, 120-200 μιη in the second week, and 200-300 μιη in the third and fourth weeks of culture.
1. - Preparación de la muestra, previa al inicio del procedimiento. 1. - Preparation of the sample, prior to the start of the procedure.
En cada punto de muestreo, en condiciones de esterilidad en cabina de flujo laminar, se retiró el medio de cultivo de un número representativo de pocilios (n = 4 a 6 pocilios de cultivo), cada uno de los cuales contenía, en promedio, entre 65 a 75 agregados celulares. Seguidamente, se realizó un doble lavado con solución tampón fosfato (500 μΙ de DPBS Sigma Aldrich Química, ref. D8537) atemperada a 37°C. A partir de este paso, los cambios de las soluciones de fijación y de etanol, se efectuaron bajo microscopio estereoscópico (Nikon SMZ 800; figura 2), con el fin de evitar la aspiración y eliminación de agregados celulares en cada pipeteo. At each sampling point, under sterile conditions in a laminar flow cabinet, the culture medium was removed from a representative number of wells (n = 4 to 6 culture wells), each of which contained, on average, between 65 to 75 cell aggregates. Then, a double wash was carried out with phosphate buffer solution (500 μΙ of DPBS Sigma Aldrich Química, ref. D8537) tempered at 37 ° C. From this step, the changes in the fixation and ethanol solutions were carried out under a stereomicroscope (Nikon SMZ 800; Figure 2), in order to avoid aspiration and elimination of cellular aggregates in each pipetting.
2. - Fijación. El sistema de fijación comprendió los siguientes pasos. Se dispensaron 500 μΙ de solución de paraformaldehído (Sigma Aldrich Química, ref. P6148) al 4% en PBS (DPBS Sigma Aldrich Química, ref. D8537) a pH 7,4, en cada pocilio de cultivo. Las muestras se dividieron en 2 grupos, de manera que la mitad de ellas se mantuvo durante 10 minutos y la otra mitad durante 15 minutos en paraformaldehído a temperatura de 2-8°C. Finalizado este periodo, se retiró la solución de fijación y se realizó un doble lavado de 5 minutos, con solución tampón fosfato (DPBS) a 2-8°C. 3. - Deshidratación en concentración creciente de etanol. 2. - Fixation. The fixing system comprised the following steps. 500 μΙ of 4% paraformaldehyde solution (Sigma Aldrich Chemistry, ref. P6148) was dispensed in PBS (DPBS Sigma Aldrich Chemistry, ref. D8537) at pH 7.4, in each culture well. The samples were divided into 2 groups, so that half of them were kept for 10 minutes and the other half for 15 minutes in paraformaldehyde at a temperature of 2-8 ° C. At the end of this period, the fixation solution was removed and a 5 minute double wash was performed, with phosphate buffer solution (DPBS) at 2-8 ° C. 3. - Dehydration in increasing concentration of ethanol.
3.1.- En la mitad de las muestras, de cada pocilio de cultivo, se retiró la solución tampón fosfato utilizada en el paso 1 y, a intervalos de 10 minutos, se dispensaron y reemplazaron, sucesivamente, 500 μΙ de etanol (Absoluto Merck Millipore, ref. 1009832500) al 30%, al 50% y al 70%, en agua ultra pura (MilliQ). Todo el proceso de deshidratación, se llevó a cabo a 2-8°C. 3.2.- La otra mitad de las muestras se deshidrató siguiendo la pauta descrita en el apartado 3.1., pero manteniendo las muestras durante 15 minutos en cada concentración de etanol. 3.1.- In half of the samples, from each culture well, the phosphate buffer solution used in step 1 was removed and, at intervals of 10 minutes, 500 μΙ of ethanol (Merck Millipore Absolute) were successively dispensed and replaced. , ref. 1009832500) at 30%, 50% and 70%, in ultra pure water (MilliQ). The entire dehydration process was carried out at 2-8 ° C. 3.2.- The other half of the samples were dehydrated following the pattern described in section 3.1., But keeping the samples for 15 minutes at each concentration of ethanol.
4. -Preinclusión en solución de agar. 4.-Preinclusion in agar solution.
El proceso de preinclusión, comprendió los pasos que se describen a continuación: The preinclusion process included the steps described below:
4.1. - Preparación de la solución de agar: se preparó una solución de agar para cultivo bacteriológico (Oxoid Ltd. Hampshire, Inglaterra, ref. LP001), al 1 ,5 % en agua destilada. La mezcla se mantuvo en agitación sobre una placa térmica hasta alcanzar los 85°C, temperatura aproximada de fusión del agar, evitando la ebullición. Su utilización comenzó cuando la solución estuvo completamente transparente. La solución se mantuvo en estado de agitación suave y permanente durante el tiempo en el que tuvo lugar el proceso de preinclusión, a una temperatura comprendida entre 82 y 100°C. 4.1. - Preparation of the agar solution: an agar solution for bacteriological culture (Oxoid Ltd. Hampshire, England, ref. LP001), 1.5% in distilled water, was prepared. The mixture was kept under stirring on a thermal plate until it reached 85 ° C, approximate melting temperature of the agar, avoiding boiling. Its use began when the solution was completely transparent. The solution was kept in a state of gentle and permanent agitation during the time in which the preinclusion process took place, at a temperature between 82 and 100 ° C.
4.2. - Preparación del molde de preinclusión y transferencia de las muestras: se preparó, como soporte plástico, un molde de polipropileno de 5 mm de diámetro y 3 mm de profundidad. Se cubrió por completo la base del molde con un volumen de 40 μΙ de solución de agar, evitando la formación de burbujas. Se transfirieron las muestras desde la solución de etanol al 70%, a la base de agar, coincidiendo con el inicio de la gelificación del mismo por descenso térmico (figura 2C, D), empleando para ello micropipetas capilares adaptadas a una jeringa. A continuación, bajo microscopio estereoscópico, se retiró el exceso de etanol acompañante de las muestras, con micropipetas capilares adaptadas a una jeringa y con hilos de papel absorbente Whatmann. Bajo microscopio estereoscópico, los agregados celulares fueron desplazados y ordenados en la zona central del lecho de agar en gelificación, o recién gelificado con ayuda de agujas de 25 G X 16 mm, evitando rasgar el agar. Finalmente, se depositó un volumen adicional de solución de agar (60- 70 μΙ), que fundió con la base y cubrió por completo las muestras (figura 1 F). El proceso de gelificación se completó introduciendo los moldes en la nevera a 2-8°C durante 4 horas. Seguidamente, las muestras se mantuvieron en el molde de polipropileno, en etanol al 70% a 2-8°C, hasta la inclusión en parafina, que tuvo lugar en un tiempo máximo de 24 horas. 4.2. - Preparation of the preinclusion mold and transfer of the samples: a polypropylene mold 5 mm in diameter and 3 mm deep was prepared as a plastic support. The mold base was completely covered with a volume of 40 μ 40 agar solution, preventing the formation of bubbles. Samples were transferred from 70% ethanol solution, to the agar base, coinciding with the beginning of gelation thereof by thermal descent (Figure 2C, D), using capillary micropipettes adapted to a syringe. Then, under stereoscopic microscope, the excess ethanol was removed from the samples, with capillary micropipettes adapted to a syringe and with Whatmann absorbent paper threads. Under stereoscopic microscopy, the cell aggregates were displaced and sorted in the central area of the agar bed in gelation, or freshly gelled with the help of 25 GX 16 mm needles, avoiding tearing the agar. Finally, an additional volume of agar solution (60-70 μΙ) was deposited, which melted with the base and completely covered the samples (Figure 1 F). The gelation process was completed by introducing the molds in the refrigerator at 2-8 ° C for 4 hours. Then, the samples were kept in the polypropylene mold, in 70% ethanol at 2-8 ° C, until inclusion in paraffin, which took place in a maximum time of 24 hours.
4.3.- Transferencia de los bloques de agar a cassettes de inclusión: finalizado el proceso de gelificación, se extrajeron los bloques de agar de los moldes, con ayuda de una aguja de 25 G X 16 mm, evitando rasgar el agar. Se transfirieron a cassettes de inclusión de rejilla con tapa (TESPA, Giessen, ref. 35396 Alemania), debidamente identificados, que se mantuvieron en etanol al 70% hasta su inclusión en parafina. 4.3.- Transfer of the agar blocks to inclusion cassettes: once the gelation process is finished, the agar blocks were removed from the molds, using a 25 G X 16 mm needle, avoiding tearing the agar. They were transferred to grid inclusion cassettes with cover (TESPA, Giessen, ref. 35396 Germany), duly identified, which were kept in 70% ethanol until they were included in paraffin.
5.- Procesado de las muestras para el análisis histológico. 5.1.- Inclusión en parafina, preparación de bloques y realización de cortes histológicos. 5.- Processing of the samples for histological analysis. 5.1.- Inclusion in paraffin, block preparation and realization of histological sections.
5.1.1.- Inclusión en parafina: los cassettes con las muestras embebidas en agar, fueron incluidos en parafina sintética plastificada con un punto de fusión de 56°C (Histo-Comp, Casa Álvarez, Madrid, España), en un procesador de tejidos de vacío automatizado (Leica ASP 300), mediante un programa que comprende los siguientes pasos: * ( )Etanol 70°: 45 minutos 5.1.1.- Inclusion in paraffin: the cassettes with the samples embedded in agar, were included in plasticized paraffin with a melting point of 56 ° C (Histo-Comp, Casa Álvarez, Madrid, Spain), in a processor Automated vacuum fabrics (Leica ASP 300), using a program that includes the following steps: * () 70 ° ethanol: 45 minutes
* Etanol 80°: 45 minutos * 80 ° ethanol: 45 minutes
* Etanol 96°: 2 x 45 minutos * 96 ° ethanol: 2 x 45 minutes
* Etanol 100°: 2 x 60 minutos * 100 ° ethanol: 2 x 60 minutes
* (2)Histo Clear: 2 x 45 minutos * (2) Histo Clear: 2 x 45 minutes
* Histo Clear: 60 minutos  * Histo Clear: 60 minutes
* Parafina: 180 minutos * Paraffin: 180 minutes
* Parafina: 180 minutos (1 ). Etanol Quimivita SA Barcelona Ref. AL110PHF.0N005L  * Paraffin: 180 minutes (1). Ethanol Quimivita SA Barcelona Ref. AL110PHF.0N005L
(2). Histo Clear. National Diagnostics, USA Ref. HS-202.  (2). Histo Clear. National Diagnostics, USA Ref. HS-202.
5.1.2. - Preparación de bloques: una vez finalizado el proceso de inclusión, se procedió a la realización manual de los bloques en una unidad formadora de bloques (Leica EG1 40H), constituida por un dispensador de parafina y una placa fría para la solidificación de los mismos. Para llevar a cabo este procedimiento, se introdujeron los cassettes en el depósito de parafina caliente y se retiró su tapa. Se seleccionaron y utilizaron moldes de dimensiones adecuadas, organizando de forma estandarizada los agregados de agar para su correcta identificación sobre una base de parafina solidificada por contacto con la superficie fría. El molde se cubrió por completo con parafina líquida y la base del cassette se utilizó como soporte del bloque, que se colocó en una placa fría (Leica EG 30) donde, una vez solidificada la parafina, fue desmoldado (figura 3). 5.1.2. - Preparation of blocks: once the inclusion process was completed, the blocks were carried out manually in a block-forming unit (Leica EG1 40H), consisting of a paraffin dispenser and a cold plate for solidification thereof . To carry out this procedure, the cassettes were introduced into the hot paraffin tank and its lid was removed. Molds of suitable dimensions were selected and used, standardizing the agar aggregates for proper identification on a solidified paraffin base by contact with the cold surface. The mold was completely covered with liquid paraffin and the cassette base was used as a support for the block, which was placed on a cold plate (Leica EG 30) where, once the paraffin solidified, it was unmold (figure 3).
5.1.3. - Cortes histológicos en microtomo: se realizaron secciones seriadas de los bloques, de 3-4 m de grosor, empleando para ello un microtomo de rotación motorizado (Leica RM2255). Los cortes se depositaron en un baño María termostatizado a 38°C para permitir que la parafina se extendiera y, posteriormente, se recuperaron en portaobjetos ionizados (SuperFrost® Plus, Menzel GMBH & Co, Alemania), para la realización de técnicas histológicas y de inmunohistoquímica (figuras 3, 4). Con el fin de visualizar y seleccionar las muestras para el estudio inmunohistoquímico, se realizó la observación directa al microscopio estereoscópico de los cortes en un portaobjetos sin teñir, lo que permitió identificar los agregados celulares inmersos en el agar. Cada 20 secciones, se obtuvo una para la tinción rutinaria con hematoxilina-eosina (figura 3D, E). Los portaobjetos con las secciones recuperadas del baño María se mantuvieron en una estufa a 38°C durante 24 horas, para el secado de los mismos previo a la realización de las técnicas de análisis. 5.1.4.- Demarcación de la muestra sobre el portaobjetos: antes del inicio de las técnicas de análisis histológico e inmunohistoquímico, previo al desparafinado de los cortes, las muestras se rodearon con una marca practicada con un lápiz de punta de diamante, lo que permitió su localización una vez eliminado el agar y la parafina, ya que a partir de ese momento no eran visibles macroscópicamente. 5.1.3. - Histological sections in microtome: serial sections of the blocks were made, 3-4 m thick, using a motorized rotation microtome (Leica RM2255). The cuts were placed in a water bath thermostatted at 38 ° C to allow the paraffin spread and subsequently recovered on slides ionised (SuperFrost ® Plus, Menzel GMBH & Co, Germany), for performing histological techniques and immunohistochemistry (figures 3, 4). With the purpose of To visualize and select the samples for the immunohistochemical study, direct observation under stereoscopic microscopy of the cuts was performed on an unstained slide, which allowed identifying the cell aggregates immersed in the agar. Every 20 sections, one was obtained for routine staining with hematoxylin-eosin (Figure 3D, E). The slides with the sections recovered from the water bath were kept in an oven at 38 ° C for 24 hours, for drying them prior to performing the analysis techniques. 5.1.4.- Demarcation of the sample on the slide: before the start of the histological and immunohistochemical analysis techniques, prior to the dewaxing of the cuts, the samples were surrounded with a mark made with a diamond-tipped pencil, which it allowed its location once the agar and paraffin were removed, since from that moment they were not macroscopically visible.
5.2.- Tinción histológica. 5.2.- Histological staining.
Se realizó en un teñidor lineal automatizado (Leica ST4040), e incluyó los siguientes pasos: It was performed on an automated linear dyeer (Leica ST4040), and included the following steps:
• Desparafinado en (1)Xilol (3 x 10 minutos) • Dewaxed in (1) Xilol (3 x 10 minutes)
• Etanol 100°: 10 minutos  • 100 ° ethanol: 10 minutes
• Etanol 96°: 10 minutos  • 96 ° ethanol: 10 minutes
· Etanol 70°: 10 minutos  70 ° ethanol: 10 minutes
• Lavado en agua: 10 minutos  • Water wash: 10 minutes
• Tinción con (2)hematoxilina de Harris: 10 minutos • Staining with (2) Harris hematoxylin: 10 minutes
• Lavado en agua: 10 minutos  • Water wash: 10 minutes
• Tinción con (3)eosina: 1 minuto • Staining with (3) eosin: 1 minute
· Etanol 70°: 1 minuto  70 ° ethanol: 1 minute
• Etanol 96°: 1 minuto  • 96 ° ethanol: 1 minute
• Etanol 100°: 1 minuto • Aclarado en xilol (3 x 1 minuto) • 100 ° ethanol: 1 minute • Rinse in xylol (3 x 1 minute)
• Montaje de las muestras con cubreobjetos empleando una resina sintética ((4)DPX) (1 ) Xilol. Panreac Química, Barcelona Ref. 21 1769.1714 • Mounting the samples with coverslips using a synthetic resin ( (4) DPX) (1) Xilol. Panreac Química, Barcelona Ref. 21 1769.1714
(2) Hematoxilina. Panreac Química, Barcelona Ref. UN 2024  (2) Hematoxylin. Panreac Química, Barcelona Ref. UN 2024
(3) Eosina. Panreac Química, Barcelona Ref. 251299.1608  (3) Eosin. Panreac Química, Barcelona Ref. 251299.1608
(4) DPX. Casa Álvarez, Madrid ref. 10-8500 Una vez solidificado el medio de montaje, se observaron las preparaciones histológicas al microscopio óptico (Microscopio Olympus BX40). Se evaluaron las características morfológicas de los agregados celulares, la afinidad y capacidad tintorial de las células, así como cuantas alteraciones se pudieron observar derivadas de un inadecuado procesamiento (crenación celular, depósitos citoplasmáticos anormales, vacuolización, tinción excesiva de la cromatina, entre otras).  (4) DPX. Alvarez House, Madrid ref. 10-8500 Once the mounting medium solidified, the histological preparations were observed under the optical microscope (Olympus BX40 Microscope). The morphological characteristics of the cell aggregates, the affinity and tintorial capacity of the cells were evaluated, as well as how many alterations could be observed derived from an inadequate processing (cell creation, abnormal cytoplasmic deposits, vacuolization, excessive chromatin staining, among others) .
Ejemplo 2.- Inmunolocalización del antígeno marcador de células en proliferación Ki67 en estructuras pseudofoliculares derivadas in vitro del cocultivo de folículos primordiales ováricos con células del estroma- intersticiales. Example 2. Immunolocalization of the marker antigen of Ki67 proliferating cells in pseudo-follicular structures derived in vitro from the co-culture of ovarian primordial follicles with stroma-interstitial cells.
El análisis se llevó a cabo sobre estructuras pluricelulares, denominadas pseudofolículos ováricos en desarrollo in vitro, que se mantuvieron en cultivo en medio definido, a 37°C, con 5% de CO2 y humedad a saturación. Estas estructuras adoptaban una morfología tridimensional aproximadamente esférica, con diámetros comprendidos entre las 70 y 280 μηι a lo largo del periodo de cultivo. La preparación de las muestras y los procedimientos de fijación, deshidratación, preagregación en matriz de agar, inclusión en parafina, preparación de bloques y cortes, se llevaron a cabo tal como se ha detallado en el apartado 5 del ejemplo 1 , utilizando tiempos de fijación de las muestras de 15 minutos y periodos de deshidratación de las mismas de 10 minutos con cada concentración de etanol. Sobre cortes histológicos de 3 μιτι, se llevó a cabo la ¡nmunolocalización del antígeno de células en proliferación Ki67. Para ello, se utilizó un anticuerpo monoclonal (Anti-Ki67, Leica Biosystems, Ref. KI67-MM1-L-CE) siguiendo el procedimiento de análisis inmunohistoquímico que se detalla a continuación. Se empleó una técnica validada por investigadores de este grupo, en el laboratorio de Histología y Anatomía Patológica del Hospital Clínico Veterinario de la Universidad Complutense de Madrid, empleando un kit comercial (Novolink Polymer Detection System® Novocastra, Leica Microsystems, Barcelona Ref.: RE-7140-K), que incluye los siguientes reactivos: The analysis was carried out on multicellular structures, called ovarian pseudo-follicles in development in vitro, which were maintained in culture in a defined medium, at 37 ° C, with 5% CO 2 and humidity at saturation. These structures adopted an approximately spherical three-dimensional morphology, with diameters between 70 and 280 μηι throughout the cultivation period. Sample preparation and fixation, dehydration, agar matrix pre-aggregation, paraffin inclusion, block and cut preparation procedures were carried out as detailed. in section 5 of Example 1, using 15-minute sample fixation times and 10-minute dehydration periods with each ethanol concentration. On histological sections of 3 μιτι, immunolocation of the Ki67 proliferating cell antigen was carried out. For this, a monoclonal antibody (Anti-Ki67, Leica Biosystems, Ref. KI67-MM1-L-CE) was used following the immunohistochemical analysis procedure detailed below. A technique validated by researchers of this group was used in the Histology and Pathology Laboratory of the Veterinary Clinical Hospital of the Complutense University of Madrid, using a commercial kit (Novolink Polymer Detection System ® Novocastra, Leica Microsystems, Barcelona Ref .: RE -7140-K), which includes the following reagents:
1. - Peroxidase Block: solución de bloqueo de la peroxidasa endógena, a base de peróxido de hidrógeno al 3%. 1. - Peroxidase Block: endogenous peroxidase blocking solution, based on 3% hydrogen peroxide.
2. - Protein Block: solución de bloqueo compuesta por caseína al 0,4% en solución salina con tampón fosfato, estabilizantes, agente tensioactivo y Bronidox L al 0,2% como conservante.  2. - Protein Block: blocking solution composed of 0.4% casein in saline solution with phosphate buffer, stabilizers, surfactant and 0.2% Bronidox L as a preservative.
3. - Post Primary Block: solución de bloqueo y potenciador de la penetración del polímero, que contiene suero animal al 10% (v/v) en solución salina tamponada con tris y ProClin™ 950 al 0,09%.  3. - Post Primary Block: blocking solution and polymer penetration enhancer, containing 10% animal serum (v / v) in tris buffered saline and 0.09% ProClin ™ 950.
4. - Novolink™ Polymer. conjugado polimérico de IgG antimurina de conejo con peroxidasa de rábano (cada uno a una concentración de 8 g/ml), que contiene suero animal al 10% (v/v) en solución salina tamponada con Tris y ProClin™ 950 al 0,09%.  4. - Novolink ™ Polymer. polymeric conjugate of rabbit anti-mouse IgG with radish peroxidase (each at a concentration of 8 g / ml), containing 10% animal serum (v / v) in saline solution buffered with Tris and 0.09 ProClin ™ 950 %.
5. - DAB Chromogen: 3,3'-diaminobenzidina al 1 ,74% peso/volumen, en solución estabilizante  5. - DAB Chromogen: 1,3'-diaminobenzidine 1.74% weight / volume, in stabilizing solution
6.- Novolink™ DAB Substrate Buffer (Polymer): solución tamponada con peróxido de hidrógeno al 0,05% y conservante.  6.- Novolink ™ DAB Substrate Buffer (Polymer): 0.05% hydrogen peroxide buffered solution and preservative.
7.- Hematoxylin: hematoxilina al 0,02%. Los pasos que comprende la técnica son los siguientes: 7.- Hematoxylin: 0.02% hematoxylin. The steps that the technique includes are the following:
1. - Desparafinado y rehidratación de las secciones (según se describe en el apartado 5.2.). 1. - Dewaxing and rehydration of the sections (as described in section 5.2.).
2. - Lavado en agua corriente (10 minutos).  2. - Wash in running water (10 minutes).
3. - Desenmascaramiento antigénico por calor con olla a presión en tampón citrato 10 mM pH 6.0 durante 2 minutos.  3. - Antigenic heat unmasking with pressure cooker in 10 mM citrate buffer pH 6.0 for 2 minutes.
4. - Enfriamiento de las secciones y lavado en agua desionizada.  4. - Cooling sections and washing in deionized water.
5. - Bloqueo de la peroxidasa endógena con Peroxidase Block (5 minutos).  5. - Endogenous peroxidase block with Peroxidase Block (5 minutes).
6. - Lavado en (1 ) TBS (2 x 5 minutos). 6. - Wash in (1) TBS (2 x 5 minutes).
7. - Incubación con Protein Block (5 minutos).  7. - Incubation with Protein Block (5 minutes).
8. - Lavado en TBS (2 x 5 minutos).  8. - Wash in TBS (2 x 5 minutes).
9. - Incubación con anti-K¡67 (1 :150 en TBS con Protein Block (50%(v/v)) durante 1 hora a temperatura de laboratorio, en oscuridad. 9. - Incubation with anti-K67 (1: 150 in TBS with Protein Block (50% (v / v)) for 1 hour at laboratory temperature, in the dark.
10. - Lavado en TBS (2 x 5 minutos). 10. - Wash in TBS (2 x 5 minutes).
1 1. - Incubación con Post Primary Block (30 minutos).  1 1. - Incubation with Post Primary Block (30 minutes).
12. - Lavado en TBS (2 x 5 minutos).  12. - Wash in TBS (2 x 5 minutes).
13. - Incubación con NovoLink™ Polymer durante 30 minutos.  13. - Incubation with NovoLink ™ Polymer for 30 minutes.
14. - Lavado en TBS (2 x 5 minutos).  14. - Wash in TBS (2 x 5 minutes).
15. - Revelado con solución de trabajo DAB (5 minutos).  15. - Developed with DAB work solution (5 minutes).
16. - Lavado en agua (5 minutos).  16. - Wash in water (5 minutes).
17. - Contrastar con Hematoxilina (3 minutos).  17. - Contrast with Hematoxylin (3 minutes).
18. - Lavado en agua (5 minutos).  18. - Washing in water (5 minutes).
19. - Deshidratación, aclarado y montaje con DPX.  19. - Dehydration, rinsing and assembly with DPX.
(1 )TBS: solución tampón tris. (1) TBS: tris buffer solution.
Los cortes se examinaron al microscopio óptico, con el fin de constatar la inmunolocalización del antígeno Ki67 y de realizar el análisis de imagen que permitía cuantificar las células en proliferación en los diferentes puntos temporales de evaluación (figura 4A-C). Ejemplo 3.- Inmunolocalización del enzima caspasa 3- activa, marcador de células que han iniciado el proceso de apoptosis, en estructuras pseudofoliculares derivadas in vitro del cocultivo de folículos primordiales ováricos con células del estroma-intersticiales. The sections were examined under an optical microscope, in order to verify the immunolocalization of the Ki67 antigen and to perform the image analysis that allowed quantifying the proliferating cells at the different time points of evaluation (Figure 4A-C). Example 3.- Immunolocalization of the enzyme caspase 3- activa, marker of cells that have started the apoptosis process, in pseudo-follicular structures derived in vitro from co-culture of ovarian primordial follicles with stroma-interstitial cells.
El análisis se llevó a cabo sobre pseudofolículos ováricos en desarrollo in vitro, en las condiciones descritas en el ejemplo 2. Como se ha descrito anteriormente, estas estructuras adoptaban una morfología tridimensional aproximadamente esférica, con diámetros comprendidos entre las 70 y 280 μιη a lo largo del periodo de cultivo. The analysis was carried out on ovarian pseudo-follicles in development in vitro, under the conditions described in example 2. As described above, these structures adopted an approximately spherical three-dimensional morphology, with diameters between 70 and 280 μιη along of the cultivation period.
La preparación de las muestras y los procedimientos de fijación, deshidratación, preagregación en matriz de agar, inclusión en parafina, preparación de bloques y cortes, se llevaron a cabo tal como se ha detallado en el ejemplo 2. Sample preparation and fixation, dehydration, agar matrix pre-aggregation, paraffin inclusion, block and cut preparation procedures were carried out as detailed in example 2.
Sobre cortes histológicos de 3 μιη, se llevó a cabo la inmunolocalización del enzima caspasa 3-activa, que incrementa su expresión y síntesis al inicio del proceso de apoptosis celular. Para ello, se utilizó un anticuerpo policlonal obtenido en conejo (Anti-caspasa 3-activa, RYD Systems, Ref. AF835) siguiendo el procedimiento de análisis inmunohistoquímico que se ha detallado en el ejemplo 2. On histological sections of 3 μιη, immunolocation of the enzyme caspase 3-active was carried out, which increases its expression and synthesis at the beginning of the process of cellular apoptosis. For this, a polyclonal antibody obtained in rabbit (Anti-caspase 3-active, RYD Systems, Ref. AF835) was used following the immunohistochemical analysis procedure detailed in example 2.
Los cortes se examinaron al microscopio óptico, con el fin de constatar la inmunolocalización del enzima caspasa 3-activa y de realizar el análisis de imagen que permitía cuantificar las células que habían iniciado el proceso de apoptosis en los diferentes puntos temporales de evaluación (figura 4D-F). The sections were examined under an optical microscope, in order to verify the immunolocalization of the 3-active caspase enzyme and to perform the image analysis that allowed quantifying the cells that had started the apoptosis process at the different time points of evaluation (Figure 4D -F).

Claims

REIVINDICACIONES
1. Procedimiento de doble inclusión para el estudio histológico y/o de localización de marcadores moleculares de especímenes pluricelulares de dimensiones microscópicas que comprende los siguientes pasos: 1. Double inclusion procedure for histological and / or localization study of molecular markers of multicellular specimens of microscopic dimensions comprising the following steps:
a) preparación de la muestra mediante lavados con solución tamponada y sin centrifugación previa;  a) sample preparation by washing with buffered solution and without prior centrifugation;
b) fijación de la muestra con un agente fijador durante un periodo de tiempo comprendido entre 10 y 15 minutos, a temperatura de 2-8°C; c) deshidratación de la muestra con concentraciones crecientes de etanol durante 10-15 minutos cada una, a una temperatura de 2-8°C; d) preinclusión de la muestra en agar sin centrifugación, transfiriendo dicha muestra desde la solución de etanol de deshidratación hasta una base de agar preparada previamente y en estado de gelificación por descenso térmico;  b) fixation of the sample with a fixing agent for a period of time between 10 and 15 minutes, at a temperature of 2-8 ° C; c) dehydration of the sample with increasing concentrations of ethanol for 10-15 minutes each, at a temperature of 2-8 ° C; d) preinclusion of the sample in agar without centrifugation, transferring said sample from the dehydration ethanol solution to a previously prepared agar base and in gelation state by thermal descent;
e) inclusión en parafina.  e) inclusion in paraffin.
2. Procedimiento según la reivindicación 1 que incluye un paso adicional f) de análisis histológico y/o de localización de marcadores moleculares. 2. Method according to claim 1 which includes an additional step f) of histological analysis and / or location of molecular markers.
3. Procedimiento según la reivindicación 2 en el que los análisis son inmunohistoquímicos. 3. Method according to claim 2 wherein the analyzes are immunohistochemical.
4. Procedimiento según cualquiera de las reivindicaciones anteriores en el que los especímenes pluricelulares tienen dimensiones comprendidas entre4. Method according to any of the preceding claims wherein the multicellular specimens have dimensions between
70 y 300 μηη. 70 and 300 μηη.
5. Procedimiento según cualquiera de las reivindicaciones anteriores en el que el tiempo de fijación de la muestra es de 15 minutos. 5. Method according to any of the preceding claims wherein the sample fixation time is 15 minutes.
6. Procedimiento según cualquiera de las reivindicaciones anteriores en el que el tiempo de deshidratación de la muestras es de 10 minutos con cada concentración de etanol. 6. The method according to any of the preceding claims wherein the dehydration time of the samples is 10 minutes with each concentration of ethanol.
7. Procedimiento según cualquiera de las reivindicaciones anteriores en el que la deshidratación de la muestra se realiza con 3 concentraciones crecientes de etanol. 7. Method according to any of the preceding claims wherein the dehydration of the sample is carried out with 3 increasing concentrations of ethanol.
8. Procedimiento según cualquiera de las reivindicaciones anteriores en el que los especímenes pluricelulares de dimensiones microscópicas se seleccionan del grupo que comprende: embriones, cuerpos embrioides, neuroesferas, derivados de diferenciación de células pluripotentes, fases larvarias o adultas de parásitos, cultivos bacterianos, agregados de células procedentes de cultivos celulares o de aspirados celulares y/o modelos animales microscópicos. Method according to any of the preceding claims, in which the multicellular specimens of microscopic dimensions are selected from the group comprising: embryos, embryoid bodies, neurospheres, derivatives of differentiation of pluripotent cells, larval or adult phases of parasites, bacterial cultures, aggregates of cells from cell cultures or cell aspirates and / or microscopic animal models.
9. Kit que comprende los elementos necesarios para procesar especímenes pluricelulares de dimensiones microscópicas mediante doble inclusión para su uso en el procedimiento definido en cualquiera de las reivindicaciones 1-8. 9. Kit comprising the elements necessary to process multicellular specimens of microscopic dimensions by double inclusion for use in the procedure defined in any of claims 1-8.
PCT/ES2014/000089 2013-05-31 2014-05-30 Double-inclusion method for the study of the histology and/or localisation of molecular markers in pluricellular specimens of microscopic dimensions WO2014191590A1 (en)

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