WO2011089293A1 - Histological method optimised for the preservation of antigenic epitopes and the cellular architecture of tissues from vertebrates - Google Patents

Histological method optimised for the preservation of antigenic epitopes and the cellular architecture of tissues from vertebrates Download PDF

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Publication number
WO2011089293A1
WO2011089293A1 PCT/ES2011/000016 ES2011000016W WO2011089293A1 WO 2011089293 A1 WO2011089293 A1 WO 2011089293A1 ES 2011000016 W ES2011000016 W ES 2011000016W WO 2011089293 A1 WO2011089293 A1 WO 2011089293A1
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samples
incubation
cryosubstitution
butanol
methanol
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PCT/ES2011/000016
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Spanish (es)
French (fr)
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WO2011089293A9 (en
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Iván DURÁN JIMÉNEZ
Leonor Santos Ruiz
Jesús Alberto SANTAMARÍA GARCÍA
José BECERRA RATIA
Manuel MARÍ BEFFA
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Universidad De Malaga
Ciber-Bbn
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Publication of WO2011089293A1 publication Critical patent/WO2011089293A1/en
Publication of WO2011089293A9 publication Critical patent/WO2011089293A9/en

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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
    • G01N1/36—Embedding or analogous mounting of samples
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention falls within the scope of Histology, and more specifically in the context of the methods for the preservation and maintenance of histological samples of vertebrates.
  • PFA paraformaldehyde
  • cryoprotection and sections to the cryostat Westerfield 2000
  • inclusion in paraffin and sections to the microtome PFA is poorly a fixative of the reactive properties of tissues, such as antigenicity or enzymatic activity.
  • fixatives of an alcoholic nature or salts such as Zn are usually used. These, however, do not offer an appropriate tissue presevation and consequently do not allow obtaining good quality histological sections.
  • the present invention involves an optimization of the methods and techniques known at present, which is achieved by combining cryosubstitution fixation (Hippe et al. 1989, Monaghan and Robertson. 1990, Quintana 1994 and Usuda et al. 1990) and the inclusion in a wax or paraffin of low melting temperature, around 40 ° C, such as Polyester Wax wax (Steedman, 1957).
  • the invention It consists of a new method with which optimal preservation of morphology and tissue reactive properties is achieved. This method respects the general shape of the sample, eliminating artifacts due to tissue retraction. Disclosure of the invention
  • the object of the present invention is a new optimized method of histological and histochemical preservation that improves the most commonly used techniques and offers an alternative to classical methods.
  • the method in question is compatible with any method of staining histological sections, such as immunolocation or in situ hybridization.
  • Cryosubstitution involves the immediate dehydration of the samples , which allows the elimination of numerous dehydration steps that can alter the integrity of the samples; Polyester wax or paraffin is soluble in alcohols, which avoids the use of aggressive solvents (for example, xylene) during dewaxing steps, being replaceable with 100 ° or 96 ° ethanol.
  • the claimed method comprises the following phases or stages:
  • the method object of the present invention comprises the use of alcoholic fixation (cryosubstitution in methanol) carried out at low temperatures, very effective for the maintenance of antigenicity.
  • Fixation comprises the treatment of the samples, properly prepared, with isopentane and methanol.
  • the embodiment of said method comprises a column support designed for the optimization of the process and which are claimed within the same invention.
  • the applicant does not know the description of any mechanism to preserve the form of cryosubstituted samples.
  • the claimed columns formed by two tubes of a material resistant to low temperatures, allow a replacement without contact with objects that prevents soft samples or with a defined arrangement from being deformed by the support.
  • the columns contain, one of them, isopentane and, the other, methanol, being the volume of Isopentane and methanol preferably proportional to the volume of the sample.
  • the columns are of variable size, conditioned by the sample size. The length of the column containing isopentane is proportional to the replacement time in free fall.
  • FIG. 1 Effects of several fixatives on tissue morphological preservation.
  • the tissues were fixed, included in Polyester Wax, and stained with hematoxylin-picrosirium.
  • a - H Cross sections of a 72h Danio embryo stained after cryosubstitution fixation (A), with PFA (B), with Bouin (C), with Zinc fixative (D) and with MAA (E).
  • FH respectively show an extension of the square region of AC.
  • the arrowheads (F) show the mesenchyme underlying the fold of the fin.
  • IJ Cross sections of an amphioxus adult fixed by cryosubstitution (I) and PFA (J). The bar measures 50 ⁇ .
  • a and B Sections of the smooth muscle of the mouse esophagus fixed with PFA (C) or by cryosubstitution (D). Both sections were shot with the antibody against Mimecan-Osteoglycine. Positive areas are stained red due to Alexa Fluor 594 fluorochrome (indirect immunoflurorescence). The nuclei are dyed blue due to Hoescht staining. The arrowhead points to a blood vessel. The asterisk indicates a muscle fiber. The bars measure 10 (CD) and 20 ⁇ (AB).
  • C and D Use of sections obtained by our method for different methodology such as immunolocation and in situ hybridization. Cross sections of regenerating fin (4 dpa). Staining with anti-Zns-5, marking scleroblasts or synthesizing cells of lepidotriquias in red (Alexa Fluor 594; C). In situ hybridization of a parallel section marking cells expressing collagen 10 (D).
  • FIG. 3 Schematic representation of a cryosubstitution protocol designed for the preservation of the three-dimensional form.
  • 1 Method for large samples.
  • 2 Method for small samples, such as a Danio rerio embryo.
  • Figure 4 Section of the anterior limb of a mouse embryo (stage 5.5) fixed by cryosubstitution and included in Polyester Wax. The section was stained with the antibody against PCNA and Pro-collagen. The nuclei were stained with Hoescht. Ways of carrying out the invention
  • Cryosubstitution columns Said columns are preferably formed by two external tubes of glass or plastic material resistant to low temperatures. Their size is variable, although they preferably have a minimum height depending on the size of the samples. On the other hand, the volume of isopentane and methanol they contain is preferably at least 10 times the volume of the sample. In a preferred embodiment the columns have a closing mechanism to prevent evaporation of isopentane and methanol.
  • an interchangeable container preferably a meshed bottom container.
  • the purpose of this container is to take the isopentane samples without residues and be able to quickly transfer it to the methanol column.
  • the pore size can be variable, provided it meets the objective of retaining the sample (s) while the isopentane is drained.
  • the interchangeable container is complemented with a slide or equivalent where the sample (s) is placed when cryosubstitute is intended to be maintained maintaining a flat shape ( Figure 3.1).
  • the sample (s) is introduced into the column containing isopentane in suspension in a drop of 70% methanol ( Figure 3.2).
  • samples with skin or epithelia for example, amphiox body, mouse embryo, or whole organs
  • samples with skin or epithelia preferably have a thickness on the millimeter scale, and more preferably a thickness less than or equal to 5 mm ; while in the case of sectioned tissues (for example, sections of the heart or brain), with greater permeability due to the absence of epithelia on their surface, larger thicknesses (for example, 1 cm) are acceptable.
  • Incubation in the isopentane column is preferably in the minute scale, and more preferably in the range of 1 to 10 minutes, and may be 1 minute sufficient for very small and permeable samples (eg, Danio rerio embryos), and being 2 minutes a valid time for a wide variety of samples.
  • the incubation is preferably in the hours scale, and more preferably in the range of 24 (very small and permeable samples) to 48 hours (valid time for a large variety of samples), although not there is a maximum incubation time, the samples can be stored in methanol at -80 ° C, and even at -20 ° C, for months.
  • Histology The following is an example, in the form of a protocol, of a preferred embodiment of the method object of the invention.
  • the isopentane and methanol columns Prior to the application of the method, the isopentane and methanol columns must be cooled to -80 ° C, and the samples to be treated must be adequately prepared (for example, decoration in the case of embryos, or dissected in the case of organs ).
  • the primary antibodies used allow the detection of epitopes of cytoplasmic membrane, extracellular matrix and intracellular organelles that usually have difficulty in antigen preservation.
  • the antibodies used for the validation of the method object of the invention are:
  • Anti-mimecan rabbit polyclonal antibody against mouse mimecan protein (Fernández et al. 2003);
  • Ki-S5 (Ki-67; a mouse polyclonal antibody, Roche);
  • Pro-collagen I (M-38; a mouse monoclonal antibody, Developmental Studies Hybridoma Bank);
  • Anti-PCNA mouse monoclonal antibody, SIGMA
  • RNA probes for the col 10a 1 gene labeled with digoxigenin were dewaxed and rehydrated in successive steps of lower grade alcohols (RNAse free). They were prehybridized in hybridization buffer for 1 hour at 70 ° C. Subsequently it hybridizes at the same temperature overnight. After washing, the sections were blocked for 4 hours in Western Bloking Reagent (Roche) according to the manufacturer's instructions and incubated in anti-digoxigenin overnight. The development was carried out by NBT / BCIP.
  • the fin folds are particularly sensitive to histological methods, usually showing ruptures between the mesenchyme and the ectoderm in most of the methods studied.
  • optimal preservation was always obtained after cryosubstitution fixation (Figure 1A).
  • the fixations with Bouin (figure IB), PFA (figure 1C), Zn fixer (figure ID) or MAA (figuralE; table 1) showed worse quality results.
  • the PFA fixation generated artefacts of mesenchymal rupture.
  • the application of cryosubstitution significantly reduces the appearance of such artifacts ( Figures 1F, 1G and 1H).
  • FIGS. II and 1J show clear histological differences between cryosubstitution methods (figure II) and fixation with PFA (figure 1J) applied to the amphioxus. While abundant discontinuities in muscle masses and loss of connective integrity are observed in samples fixed with PFA, cryosubstituted samples have no morphological defect.
  • FIGS 2 A and B show stains with an antibody against mimechan of the smooth muscle of the mouse esophageal wall fixed with PFA (Figure 2A) or by cryosubstitution ( Figure 2B). While the section fixed with paraformaldehydes shows very low immuno-reactivity, the section obtained after cryosubstitution shows a very strong and specific immuno-staining of muscle fibers and blood vessels.
  • Figures 2C and D show parallel cross sections of regenerative fin of 4 days post-amputation (dpa) obtained by our method.
  • Figure 2C shows an immuno fluorescence staining of Zns-5.
  • Lepidotriquia is a skeletal structure of the fin that has markers of both bone and cartilage. This can be demonstrated by in situ hybridization of collagen messenger RNA 10 ( Figure 2D). In this case, the parallel sections allow both markers to be located in the same cells using such different methodologies. Therefore, this methodology allows the preservation of the antigenicity of epitopes while maintaining the integrity of messenger RNAs so sensitive to degradation by AR handles.
  • cryosubstitution columns also allows the shape and relative position of the various tissues within the samples to be preserved. This method allows the tissue to be preserved from possible folding by manipulation (for example, keeping an extended fin; figure 3.1).
  • Another common problem in small-sized samples is significant shape transformations following classical cryosubstitution methods. An example of this is the embryos of Danio rerio, who, when cryosubstituted on a surface, suffer deformations due to the compression of the tissues against the support.
  • the drop of a drop of diluted alcohol in the isopentane column allows homogeneous substitution over the entire surface of the sample avoiding deformation ( Figure 3.2).
  • the method object of the present invention has also proven to be very useful for obtaining histological sections of samples that intersperse soft and hard tissues.
  • the separation of both usually occurs as an artifact during dehydration-rehydration phenomena or while being cut to the cryostat or microtome.
  • the direct and rapid replacement of the water content during cryosubstitution and the rigidity that this wax or paraffin contributes to soft tissues solve these problems.
  • An example is represented in cartilage samples, which are surrounded by connective tissues such as the trachea, or the mouse limb during development ( Figure 4).
  • the claimed method is useful for analyzing samples stained with histochemical methods, or with fluorescent labels such as endogenous fluorescence of transgenic organisms.
  • the fluorescent properties of the tissues are maintained throughout the histological processing due to the low temperature used during fixation and inclusion.
  • the method object of the present invention improves most of the current histological methods designed for optical microscopy.
  • This method also improves the antigenic properties of embryonic and adult tissues of amphioxus, Danio rerio and mouse, preserving their structure and preventing the degradation of RNA.
  • This method is a good alternative to the classical histology methods normally used in embryogenesis studies of vertebrates in the face of such common problems as epitope lability or intercalation of rigid tissues in soft tissues. References

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Abstract

Histological method optimised for the preservation of antigenic epitopes and the cellular architecture of tissues from vertebrates, characterised in that it combines fixing through cryosubstitution and inclusion in a low melting point wax or paraffin; the method of the present invention improves the majority of the present histological methods designed for optical microscopy. Said method furthermore improves the antigenic properties of amphioxus, Danio rerio and rat embryonic and adult tissues, preserving the structure thereof and preventing RNA degradation. This method constitutes a good alternative to classic histological methods normally utilised in embryogenesis studies of vertebrates in respect of problems as common as the lability of epitopes or the intercalation of rigid tissues in soft tissues.

Description

Título  Title
Método histológico optimizado para la preservación de epítopos antigénicos y de la arquitectura celular de tejidos de vertebrados Optimized histological method for the preservation of antigenic epitopes and the cellular architecture of vertebrate tissues
Sector técnico Technical sector
La presente invención se encuadra en el ámbito de la Histología, y más concretamente en el contexto de los métodos para la preservación y el mantenimiento de muestras histológicas de vertebrados. The present invention falls within the scope of Histology, and more specifically in the context of the methods for the preservation and maintenance of histological samples of vertebrates.
Técnica anterior Prior art
La preservación y el mantenimiento de la arquitectura celular y de la antigenicidad nativa durante el procesado histológico es un reto continuo de la Biología Celular. Durante los últimos diez años, se ha desarrollado el estudio de un buen número de organismos modelos útiles para el análisis experimental de muchos aspectos de la biología de los vertebrados, incluido su desarrollo (Haffter et al. 1996; Capdevila et al. 2000). La Biología del Desarrollo y otras disciplinas también requieren técnicas histológicas que permitan localizar tanto ARN mensajeros como proteínas en experimentos dirigidos a conocer la regulación y función génicas. The preservation and maintenance of cellular architecture and native antigenicity during histological processing is a continuing challenge of Cellular Biology. During the last ten years, the study of a number of useful model organisms for the experimental analysis of many aspects of vertebrate biology has been developed, including its development (Haffter et al. 1996; Capdevila et al. 2000). Developmental Biology and other disciplines also require histological techniques that allow to locate both messenger RNAs and proteins in experiments aimed at knowing gene regulation and function.
Las técnicas actuales más usadas son la fijación mediante paraformaldehido (PFA) seguida de crioprotección y secciones al criostato (Westerfield 2000) o inclusión en parafina y secciones al microtomo. Sin embargo, el PFA es mal un fijador de las propiedades reactivas de los tejidos, tales como la antigenicidad o la actividad enzimática.  The most commonly used current techniques are fixation by paraformaldehyde (PFA) followed by cryoprotection and sections to the cryostat (Westerfield 2000) or inclusion in paraffin and sections to the microtome. However, PFA is poorly a fixative of the reactive properties of tissues, such as antigenicity or enzymatic activity.
En los casos de epítopos sensibles a los métodos más comunes de fijación se suelen usar fijadores de naturaleza alcohólica o de sales como el Zn. Estos, sin embargo, no ofrecen una apropiada presevación tisular y consecuentemente no permiten la obtención de secciones histológicas de buena calidad.  In cases of epitopes sensitive to the most common methods of fixation, fixatives of an alcoholic nature or salts such as Zn are usually used. These, however, do not offer an appropriate tissue presevation and consequently do not allow obtaining good quality histological sections.
La presente invención supone una optimización de los métodos y técnicas conocidos en la actualidad, lo que se consigue mediante la combinación de la fijación por criosustitución (Hippe et al. 1989, Monaghan and Robertson. 1990, Quintana 1994 and Usuda et al. 1990) y la inclusión en una cera o parafina de baja temperatura de fusión, en torno a 40 °C, como por ejemplo la cera Polyester Wax ( Steedman, 1957). La invención consiste en un método nuevo con el cual se logra una preservación óptima de la morfología y de las propiedades reactivas del tejido. Este método respeta la forma general de la muestra, eliminando los artefactos debidos a la retracción tisular. Divulgación de la invención The present invention involves an optimization of the methods and techniques known at present, which is achieved by combining cryosubstitution fixation (Hippe et al. 1989, Monaghan and Robertson. 1990, Quintana 1994 and Usuda et al. 1990) and the inclusion in a wax or paraffin of low melting temperature, around 40 ° C, such as Polyester Wax wax (Steedman, 1957). The invention It consists of a new method with which optimal preservation of morphology and tissue reactive properties is achieved. This method respects the general shape of the sample, eliminating artifacts due to tissue retraction. Disclosure of the invention
El objeto de la presente invención es un nuevo método optimizado de preservación histológica e histoquímica que mejora las técnicas más utilizadas actualmente y ofrece una alternativa a los métodos clásicos. El método en cuestión es compatible con cualquier método de tinción de secciones histológicas, como la inmunolocalización o la hibridación in situ. The object of the present invention is a new optimized method of histological and histochemical preservation that improves the most commonly used techniques and offers an alternative to classical methods. The method in question is compatible with any method of staining histological sections, such as immunolocation or in situ hybridization.
Por último, el proceso de fijación se combina con materiales de inclusión óptimos para la integridad tisular a la vez que sigue preservando la antigenicidad natural del tejido y reduce pasos del protocolo que aumentan la eficacia del proceso: La criosustitución implica la deshidratación inmediata de las muestras, lo que permite la eliminación de numerosos pasos de deshidratación que pueden alterar la integridad de las muestras; la cera o parafina de poliéster es soluble en alcoholes, lo que permite evitar el uso de solventes agresivos (por ejemplo, xileno) durante los pasos de desparafinado, siendo sustituibles por etanol 100 ° ó 96 °.  Finally, the fixation process is combined with optimal inclusion materials for tissue integrity while still preserving the natural antigenicity of the tissue and reducing protocol steps that increase the effectiveness of the process: Cryosubstitution involves the immediate dehydration of the samples , which allows the elimination of numerous dehydration steps that can alter the integrity of the samples; Polyester wax or paraffin is soluble in alcohols, which avoids the use of aggressive solvents (for example, xylene) during dewaxing steps, being replaceable with 100 ° or 96 ° ethanol.
El método reivindicado comprende las siguientes fases o etapas:  The claimed method comprises the following phases or stages:
Fijación por criosustitución Cryosubstitution fixation
El método objeto de la presente invención comprende el uso de fijación alcohólica (criosustitución en metanol) efectuada a bajas temperaturas, muy efectivo para el mantenimiento de la antigenicidad. La fijación comprende el tratamiento de las muestras, adecuadamente preparadas, con isopentano y metanol. The method object of the present invention comprises the use of alcoholic fixation (cryosubstitution in methanol) carried out at low temperatures, very effective for the maintenance of antigenicity. Fixation comprises the treatment of the samples, properly prepared, with isopentane and methanol.
La realización de dicho método comprende un soporte en columnas diseñado para la optimización del proceso y que son reivindicadas dentro de la misma invención. El solicitante no conoce la descripción de ningún mecanismo para preservar la forma de muestras criosustituidas. Las columnas reivindicadas, formadas por dos tubos de un material resistente a bajas temperaturas, permiten una sustitución sin contacto con objetos que evita que muestras blandas o con disposición definida se deformen por el soporte. Las columnas contienen, una de ellas, isopentano y, la otra, metanol, siendo el volumen de isopentano y de metanol preferentemente proporcional al volumen de la muestra. Las columnas son de tamaño variable, condicionado por el tamaño de la muestra. La longitud de la columna que contiene isopentano es proporcional el tiempo de sustitución en caída libre. Durante el hundimiento de la muestra, todo el contenido hídrico es sustituido por isopentano y, paralelamente, la muestra se congela. Por ello, el que este proceso ocurra durante la caída sin contacto con ningún instrumento, permite que la sustitución sea homogénea a través de toda su superficie y no se pierda la forma. Finalmente, la muestra llega al fondo, rígida por la congelación y deshidratada. Tras el tiempo requerido, la muestra es trasladada a la segunda columna, con metanol. The embodiment of said method comprises a column support designed for the optimization of the process and which are claimed within the same invention. The applicant does not know the description of any mechanism to preserve the form of cryosubstituted samples. The claimed columns, formed by two tubes of a material resistant to low temperatures, allow a replacement without contact with objects that prevents soft samples or with a defined arrangement from being deformed by the support. The columns contain, one of them, isopentane and, the other, methanol, being the volume of Isopentane and methanol preferably proportional to the volume of the sample. The columns are of variable size, conditioned by the sample size. The length of the column containing isopentane is proportional to the replacement time in free fall. During the sinking of the sample, all the water content is replaced by isopentane and, in parallel, the sample is frozen. Therefore, the fact that this process occurs during the fall without contact with any instrument, allows the replacement to be homogeneous throughout its surface and the shape is not lost. Finally, the sample reaches the bottom, rigid by freezing and dehydrated. After the required time, the sample is transferred to the second column, with methanol.
Inclusión en parafina de temperatura de fusión baja Paraffin inclusion of low melting temperature
Comprende la incubación de las muestras fijadas en diferentes soluciones (metanol :butanol 1 : 1, butanol, butanohcera poliéster 1 : 1) a diferentes temperaturas, que van desde temperatura ambiente hasta 40 °C; y su solidificación en parafina poliéster. It includes the incubation of the samples fixed in different solutions (methanol: butanol 1: 1, butanol, butanohcera polyester 1: 1) at different temperatures, ranging from room temperature to 40 ° C; and its solidification in polyester paraffin.
Obtención de secciones Obtaining sections
Comprende el uso de microtomo. Understand the use of microtome.
Montaje Mounting
Comprende el uso de agua destilada estéril. Descripción de las figuras It includes the use of sterile distilled water. Description of the figures
Figura 1. Efectos de varios fijadores en la preservación morfológica de los tejidos. Los tejidos fueron fijados, incluidos en Polyester Wax, y teñidos con la hematoxilina- picrosirio. A - H Secciones transversales de un embrión de Danio rerio de 72h teñidas tras fijación por criosustitución (A), con PFA (B), con Bouin (C), con fijador de Zinc (D) y con MAA (E). F-H muestran respectivamente una ampliación de la región cuadrada de A-C. Las puntas de flecha (F) muestran el mesénquima subyacente al pliegue de la aleta. I-J: Secciones transversales de un adulto de anfioxo fijado por criosustitución (I) y PFA (J). La barra mide 50 μηι. Figura 2. Efecto de la fijación en la preservación antigénica. A y B: Secciones del músculo liso del esófago del ratón fijadas con PFA (C) o por criosustitución (D). Ambas secciones se tifleron con el anticuerpo contra Mimecán-Osteoglicina. Las áreas positivas se muestran teñidas de rojo debido al fluorocromo Alexa Fluor 594 (inmunoflurorescencia indirecta). Los núcleos se muestran teñidos de azul debido a la tinción Hoescht. La punta de flecha señala un vaso sanguíneo. El asterisco señala una fibra muscular. Las barras miden 10 (C-D) y 20 μηι (A-B). C y D: Uso de secciones obtenidas por nuestro método para diferentes metodología como la inmunolocalización e hibridación in situ. Secciones transversales de aleta regenerante (4 dpa). Tinción con anti-Zns-5, marcando escleroblastos o células sintetizadoras de lepidotriquias en rojo (Alexa Fluor 594; C). Hibridación in situ de una sección paralela marcando células que expresan colágeno 10 (D). Figure 1. Effects of several fixatives on tissue morphological preservation. The tissues were fixed, included in Polyester Wax, and stained with hematoxylin-picrosirium. A - H Cross sections of a 72h Danio embryo stained after cryosubstitution fixation (A), with PFA (B), with Bouin (C), with Zinc fixative (D) and with MAA (E). FH respectively show an extension of the square region of AC. The arrowheads (F) show the mesenchyme underlying the fold of the fin. IJ: Cross sections of an amphioxus adult fixed by cryosubstitution (I) and PFA (J). The bar measures 50 μηι. Figure 2. Effect of fixation on antigen preservation. A and B: Sections of the smooth muscle of the mouse esophagus fixed with PFA (C) or by cryosubstitution (D). Both sections were shot with the antibody against Mimecan-Osteoglycine. Positive areas are stained red due to Alexa Fluor 594 fluorochrome (indirect immunoflurorescence). The nuclei are dyed blue due to Hoescht staining. The arrowhead points to a blood vessel. The asterisk indicates a muscle fiber. The bars measure 10 (CD) and 20 μηι (AB). C and D: Use of sections obtained by our method for different methodology such as immunolocation and in situ hybridization. Cross sections of regenerating fin (4 dpa). Staining with anti-Zns-5, marking scleroblasts or synthesizing cells of lepidotriquias in red (Alexa Fluor 594; C). In situ hybridization of a parallel section marking cells expressing collagen 10 (D).
Figura 3. Representación esquemática de un protocolo de criosustitución diseñado para la preservación de la forma tridimensional. 1 : Método para muestras grandes. 2: Método para muestras de tamaño pequeño, como podría ser un embrión de Danio rerio.  Figure 3. Schematic representation of a cryosubstitution protocol designed for the preservation of the three-dimensional form. 1: Method for large samples. 2: Method for small samples, such as a Danio rerio embryo.
Figura 4. Sección de la extremidad anterior de un embrión de ratón (estadio El 5.5) fijado por criosustitución e incluido en Polyester Wax. La sección fue teñida con el anticuerpo contra la PCNA y el Pro-colágeno. Los núcleos se tiñeron con Hoescht. Maneras de realización de la invención  Figure 4. Section of the anterior limb of a mouse embryo (stage 5.5) fixed by cryosubstitution and included in Polyester Wax. The section was stained with the antibody against PCNA and Pro-collagen. The nuclei were stained with Hoescht. Ways of carrying out the invention
Para validar el método objeto de la presente invención se han utilizado embriones y tejidos adultos de varias especies de cordados, numerosos anticuerpos primarios diferentes, específicos de diversos epítopos, y varias sondas génicas que han permitido conocer la localización de diversos ARN mensajeros de las distintas especies utilizadas. Los anticuerpos usados en las pruebas de optimización fueron marcados tanto con marcadores fluorescentes como con enzimas. Algunos anticuerpos específicos de epítopos muy lábiles, y que habían dado muchos problemas con las técnicas anteriores, ahora se muestran eficaces a la hora de detectar la localización de los antígenos. In order to validate the method object of the present invention, adult embryos and tissues of several species of chordates, numerous different primary antibodies, specific of various epitopes, and several gene probes that have allowed to know the location of various messenger RNAs of the different species used The antibodies used in the optimization tests were labeled with both fluorescent markers and enzymes. Some specific antibodies of very labile epitopes, and that had given many problems with the prior techniques, are now shown to be effective in detecting the location of the antigens.
A continuación se procede a describir, sin carácter limitativo, condiciones y variables que permiten una realización preferida y óptima del método objeto de la presente invención.  Next, we proceed to describe, without limitation, conditions and variables that allow a preferred and optimal embodiment of the method object of the present invention.
Columnas de criosustitución: Dichas columnas están preferentemente formadas por dos tubos externos de vidrio o material plástico resistente a bajas temperaturas. Su tamaño es variable, aunque preferentemente presentan una altura mínima dependiente del tamaño de las muestras. Por otra parte, el volumen de isopentano y de metanol que contienen es preferentemente de al menos 10 veces el volumen de la muestra. En una realización preferida las columnas presentan un mecanismo de cierre para evitar la evaporación del isopentano y del metanol. Cryosubstitution columns: Said columns are preferably formed by two external tubes of glass or plastic material resistant to low temperatures. Their size is variable, although they preferably have a minimum height depending on the size of the samples. On the other hand, the volume of isopentane and methanol they contain is preferably at least 10 times the volume of the sample. In a preferred embodiment the columns have a closing mechanism to prevent evaporation of isopentane and methanol.
Para el traspaso de la/s muestra/s de una columna a otra, se usa un recipiente intercambiable, preferentemente un recipiente de fondo mallado. El objeto de este recipiente es el de sacar la/s muestras del isopentano sin restos de éste y poder pasarlo rápidamente a la columna de metanol. El tamaño del poro puede ser variable, siempre que cumpla con el objetivo de retener la/s muestra/s mientras se escurre el isopentano. En otra realización preferida, el recipiente intercambiable se complementa con un portaobjetos o equivalente donde se coloca/n la/s muestra/s cuando se pretenda criosustituir tejidos manteniendo una forma plana (figura 3.1). En otra realización preferida, la/s muestra/s se introduce/n en la columna que contiene isopentano en suspensión en una gota de metanol al 70% (figura 3.2).  For the transfer of the sample (s) from one column to another, an interchangeable container, preferably a meshed bottom container, is used. The purpose of this container is to take the isopentane samples without residues and be able to quickly transfer it to the methanol column. The pore size can be variable, provided it meets the objective of retaining the sample (s) while the isopentane is drained. In another preferred embodiment, the interchangeable container is complemented with a slide or equivalent where the sample (s) is placed when cryosubstitute is intended to be maintained maintaining a flat shape (Figure 3.1). In another preferred embodiment, the sample (s) is introduced into the column containing isopentane in suspension in a drop of 70% methanol (Figure 3.2).
Tamaño de las muestras. Sample size
El tamaño admisible de las muestras a fijar mediante criosustitución viene determinado por su permeabilidad. De este modo, es recomendable que las muestras con piel o epitelios (por ejemplo, cuerpo de anfioxo, embrión de ratón, u órganos completos) tengan preferentemente un grosor en la escala de milímetros, y más prefentemente un grosor menor o igual a 5 mm; mientras que en el caso de tejidos seccionados (por ejemplo, secciones de corazón o cerebro), con mayor permeabilidad debido a la inexistencia de epitelios en sus superficie, son aceptables grosores mayores (por ejemplo, 1 cm). The permissible size of the samples to be fixed by cryosubstitution is determined by their permeability. Thus, it is recommended that samples with skin or epithelia (for example, amphiox body, mouse embryo, or whole organs) preferably have a thickness on the millimeter scale, and more preferably a thickness less than or equal to 5 mm ; while in the case of sectioned tissues (for example, sections of the heart or brain), with greater permeability due to the absence of epithelia on their surface, larger thicknesses (for example, 1 cm) are acceptable.
Tiempos de criosustitución: Cryosubstitution times:
Dichos tiempos dependen del tamaño y de la permaibilidad de las muestras. La incubación en la columna de isopentano es preferentemente en la escala de minutos, y más preferentemente en el rango de 1 a 10 minutos, pudiendo ser 1 minuto suficiente para muestras muy pequeñas y permeables (por ejemplo, embriones de Danio rerio), y siendo 2 minutos un tiempo válido para una gran variedad de muestras. En el caso de la columna de metanol, la incubación es preferentemente en la escala de horas, y más preferentemente en el rango de 24 (muestras muy pequeñas y permeables) a 48 horas (tiempo válido para una gran variedad de muestras), aunque no existe un tiempo máximo de incubación, pudiendo ser almacenadas las muestras en metanol a -80 °C, e incluso a -20 °C, durante meses. These times depend on the size and permability of the samples. Incubation in the isopentane column is preferably in the minute scale, and more preferably in the range of 1 to 10 minutes, and may be 1 minute sufficient for very small and permeable samples (eg, Danio rerio embryos), and being 2 minutes a valid time for a wide variety of samples. In the case of the methanol column, the incubation is preferably in the hours scale, and more preferably in the range of 24 (very small and permeable samples) to 48 hours (valid time for a large variety of samples), although not there is a maximum incubation time, the samples can be stored in methanol at -80 ° C, and even at -20 ° C, for months.
Tejidos estudiados El método se ha aplicado a embriones y tejidos adultos de anfioxo, de Danio revio, y ratón. Los embriones de pez (D. rerió) se obtuvieron a través de cruces entre individuos comprados en tiendas de la ciudad y mantenidos a 25 °C en acuarios con aireación. Los embriones de ratón se obtuvieron mediante cruzamiento de adultos del Estabularlo de la Universidad de Málaga. Los especímenes adultos del anfioxo se obtuvieron en la localidad francesa de Banyuls. La manipulación animal respetó la normativa establecida en el B.O.E. 67, 1988. Tissues studied The method has been applied to embryos and adult tissues of amphioxus, Danio revio, and mouse. Fish embryos (D. laughed) were obtained through crossings between individuals bought in stores in the city and kept at 25 ° C in aerated aquariums. Mouse embryos were obtained by crossing adults from the Establish it of the University of Malaga. The adult amphiox specimens were obtained in the French town of Banyuls. Animal handling complied with the regulations established in the B.O.E. 67, 1988.
Histología A continuación se refiere un ejemplo, en forma de protocolo, de realización preferida del método objeto de la invención. Histology The following is an example, in the form of a protocol, of a preferred embodiment of the method object of the invention.
Previo a la aplicación del método, las columnas de isopentano y de metanol deben ser enfriadas a -80 °C, y las muestras a tratar deben ser adecuadamente preparadas (por ejemplo, decorionación en el caso de embriones, o disecado en el caso de órganos).  Prior to the application of the method, the isopentane and methanol columns must be cooled to -80 ° C, and the samples to be treated must be adequately prepared (for example, decoration in the case of embryos, or dissected in the case of organs ).
Fijación por criosustitución Cryosubstitution fixation
1. Transferir las muestras al recipiente intercambiable introducido en la columna de criosustitución que contiene isopentano enfriado a -80 °C (figura 3 A). Dicha transferencia puede realizarse con la ayuda de un portaobjetos o equivalente en el que se ha colocado la muestra (figura 3.1), en suspensión en metanol al 70%, o simplemente dejando caer la muestra en su interior (figura 3.2). 2. Incubar las muestras en isopentano (figura 3A) durante un tiempo determinado en función del tamaño y de la permeabilidad de la muestras. 1. Transfer the samples to the exchangeable container introduced in the cryosubstitution column containing isopentane cooled to -80 ° C (Figure 3 A). Said transfer can be made with the help of a slide or equivalent in which the sample has been placed (figure 3.1), suspended in 70% methanol, or simply by dropping the sample inside (figure 3.2). 2. Incubate the samples in isopentane (Figure 3A) for a certain time depending on the size and permeability of the samples.
3. Transferir el recipiente intercambiable con las muestras a la columna de metanol enfriado a -80 °C (figura 3B).  3. Transfer the exchangeable container with the samples to the methanol column cooled to -80 ° C (Figure 3B).
4. Incubar las muestras en metanol (figura 3B) durante un tiempo determinado en función del tamaño y de la permeabilidad de la muestras y continuar con la inclusión en cera o parafina de temperatura de fusión baja, o directamente almacenar las muestras en metanol a -80 "C o a -20 °C (continuar con la inclusión requerirá, en el caso de que las muestras sean almacenadas, su recuperación mediante la incubación de las muestras en un gradiente de temperaturas desde -80 °C ó -20 °C hasta temperatura ambiente).  4. Incubate the samples in methanol (Figure 3B) for a certain time depending on the size and permeability of the samples and continue with the inclusion in wax or paraffin of low melting temperature, or directly store the samples in methanol at - 80 "C or -20 ° C (continuing with the inclusion will require, in the event that the samples are stored, their recovery by incubating the samples in a temperature gradient from -80 ° C or -20 ° C to temperature ambient).
Inclusión en cera o parafina de temperatura de fusión baja Inclusion in wax or paraffin of low melting temperature
1. Incubar las muestras a temperatura ambiente en una mezcla 1 : 1 de metanol y butanol durante 15 - 45 minutos, en función del tipo de muestra; durante 30 minutos en una realización más preferida, apta para una gran variedad de muestras. 1. Incubate the samples at room temperature in a 1: 1 mixture of methanol and butanol for 15-45 minutes, depending on the type of sample; for 30 minutes in a more preferred embodiment, suitable for a wide variety of samples.
2. Incubar en butanol a temperatura ambiente durante 5 - 20 minutos, en función del tipo de muestra; durante 10 minutos en una realización más preferida, apta para una gran variedad de muestras.  2. Incubate in butanol at room temperature for 5-20 minutes, depending on the type of sample; for 10 minutes in a more preferred embodiment, suitable for a wide variety of samples.
3. Incubar en butanol a 40 °C durante 10 minutos.  3. Incubate in butanol at 40 ° C for 10 minutes.
4. Incubar en una mezcla de butanol y cera o parafina poliéster (1 :1) durante 30 minutos a 40 °C.  4. Incubate in a mixture of butanol and wax or polyester paraffin (1: 1) for 30 minutes at 40 ° C.
5. Incubar en cera o parafina poliéster tres veces a 40 °C durante 30 minutos cada vez. 5. Incubate in wax or polyester paraffin three times at 40 ° C for 30 minutes each time.
6. Colocar la cera o parafina poliéster sobre el molde. 6. Place the wax or polyester paraffin on the mold.
7. Solidificar completamente el bloque de cera o parafina, con la muestra en su interior, a 4 °C. Obtención de secciones 7. Completely solidify the wax or paraffin block, with the sample inside, at 4 ° C. Obtaining sections
Cortar las secciones al microtomo, preferentemente en un lugar climatizado, y almacenar las mismas a 4 °C hasta su montaje. Cut the sections to the microtome, preferably in a heated place, and store them at 4 ° C until assembly.
Montaje de secciones Section Assembly
Usar agua destilada estéril, preferentemente agua bidestilada estéril enfriada a 4 °C. Validación Use sterile distilled water, preferably sterile double-distilled water cooled to 4 ° C. Validation
Como métodos de control y validación del método se han utilizado paraformaldehido (Akimenko et al. 1995), fijador de Bouin (Marí-Beffa et al. 1996), fijador de Zn (Beckstead 1994), fijador MAA (metanol:acetona:agua 1 : 1 :8; Mufloz-Chápuli et al. 1997); y la inclusión en parafina Histosec (Murciano et al. 2002). El resultado de la comparación entre el método objeto de la presente invención y los métodos de referencia antes citados es el siguiente (tabla 1): Paraformaldehyde (Akimenko et al. 1995), Bouin fixative (Marí-Beffa et al. 1996), Zn fixative (Beckstead 1994), MAA fixer (methanol: water 1) have been used as control and validation methods. : 1: 8; Mufloz-Chápuli et al. 1997); and inclusion in Histosec paraffin (Murciano et al. 2002). The result of the comparison between the method object of the present invention and the reference methods mentioned above is as follows (table 1):
Figure imgf000010_0001
Figure imgf000010_0001
(+ muestra grados de preservación tisular y antigénica. +++ = preservación muy alta, - = preservación muy pobre, * indica resultados muy variables dependiendo del tejido estudiado)  (+ shows degrees of tissue and antigen preservation. +++ = very high preservation, - = very poor preservation, * indicates very variable results depending on the tissue studied)
Tinciones con anticuerpos e histoquímica Staining with antibodies and histochemistry
Los anticuerpos primarios utilizados permiten la detección de epítopos de membrana citoplasmática, matriz extracelular y orgánulos intracelulares que suelen presentar dificultad de preservación antigénica. Los anticuerpos utilizados para la validación del método objeto de la invención son: The primary antibodies used allow the detection of epitopes of cytoplasmic membrane, extracellular matrix and intracellular organelles that usually have difficulty in antigen preservation. The antibodies used for the validation of the method object of the invention are:
1. Flg (anticuerpo policlonal contra el dominio intra-membrana del FGFR1), 2. Bek C17 (anticuerpo policlonal contra el dominio intra-membrana del FGFR2), 1. Flg (polyclonal antibody against the intra-membrane domain of FGFR1), 2. Bek C17 (polyclonal antibody against the intra-membrane domain of FGFR2),
3. C15 (anticuerpo policlonal contra el dominio intra-membrana del FGFR3) y 3. C15 (polyclonal antibody against the intra-membrane domain of FGFR3) and
4. C16 (anticuerpo policlonal contra el dominio intra-membrana del FGFR4; los cuatro obtenidos por Santa Cruz Biotechnology, Inc. Heidelberg, Alemania); 4. C16 (polyclonal antibody against the intra-membrane domain of FGFR4; the four obtained by Santa Cruz Biotechnology, Inc. Heidelberg, Germany);
5. Anti-mimecán (anticuerpo policlonal de conejo contra la proteína mimecán de ratón (Fernández et al. 2003);  5. Anti-mimecan (rabbit polyclonal antibody against mouse mimecan protein (Fernández et al. 2003);
6. Ki-S5 (Ki-67; un anticuerpo policlonal de ratón, Roche);  6. Ki-S5 (Ki-67; a mouse polyclonal antibody, Roche);
7. Pro-colágeno I (M-38; un anticuerpo monoclonal de ratón, Developmental Studies Hybridoma Bank);  7. Pro-collagen I (M-38; a mouse monoclonal antibody, Developmental Studies Hybridoma Bank);
8. C20 TRT (un anticuerpo policlonal de cabra, Santa Cruz Biotechnology). 8. C20 TRT (a goat polyclonal antibody, Santa Cruz Biotechnology).
9. Anti-PCNA (anticuerpo monoclonal de ratón, SIGMA) 9. Anti-PCNA (mouse monoclonal antibody, SIGMA)
Asimismo se han utilizado anticuerpos secundarios marcados con Alexa Fluor 594 (Invitrogen. Molecular Probes) o biotinilados (a una concentración 1 :2000). Estos últimos fueron detectados con la peroxidasa ExtrAvidina (Sigma) y la reacción de la peroxidasa revelada con diamino-bencidina (DAB; Beckstead 1994). Las tinciones hematoxilina- picrosirio (HP) y Hoescht se utilizaron como técnicas histoquímicas de modo simple o acompañando las tinciones con anticuerpos (Bechara et al. 2000). Secondary antibodies labeled with Alexa Fluor 594 (Invitrogen. Molecular Probes) or biotinylated (at a concentration of 1: 2000) have also been used. The latter were detected with the ExtrAvidin peroxidase (Sigma) and the reaction of the peroxidase revealed with diamino-benzidine (DAB; Beckstead 1994). The hematoxylin-pyrosirio (HP) and Hoescht stains were used as histochemical techniques simply or by accompanying antibody stains (Bechara et al. 2000).
Hibridación in situ In situ hybridization
La hibridación in situ se realizó según Akimenko et al. (1994) usando sondas de RNA para el gen col 10a 1 marcadas con digoxigenina. Brevemente, las secciones fueron desparafinadas y rehidratadas en pasos sucesivos de alcoholes de menor graduación (libre de RNAsa). Se prehibridaron en tampón de hibridación durante 1 hora a 70 °C. Posteriormente se híbrido a la misma temperatura durante toda la noche. Tras los lavados, las secciones se bloquearon durante 4 horas en Western Bloking Reagent (Roche) según las instrucciones del fabricante y se incubaron en anti-digoxigenina toda la noche. El revelado se llevo a cabo mediante NBT/BCIP. In situ hybridization was performed according to Akimenko et al. (1994) using RNA probes for the col 10a 1 gene labeled with digoxigenin. Briefly, the sections were dewaxed and rehydrated in successive steps of lower grade alcohols (RNAse free). They were prehybridized in hybridization buffer for 1 hour at 70 ° C. Subsequently it hybridizes at the same temperature overnight. After washing, the sections were blocked for 4 hours in Western Bloking Reagent (Roche) according to the manufacturer's instructions and incubated in anti-digoxigenin overnight. The development was carried out by NBT / BCIP.
Resultados La criosustitución en metanol, combinada con inclusión en cera o parafina de bajo punto de fusión y corte de secciones al micrótomo, parece ser una buena alternativa a la fijación con paraformaldehido (PFA), y secciones al criostato o tras inclusión en parafina tradicional. La preservación de la antigenicidad y morfología tisular se muestran excluyentes en los protocolos histológicos rutinarios. En un primer experimento, se tifleron secciones de la región caudal de embriones de Danio re io con hematoxilina-picrosirio. En estos estadios, los miotomos y los pliegues de aletas dorsal y anal están bien desarrollados. Los pliegues de la aleta son particularmente sensibles a los métodos histológicos, mostrando normalmente rupturas entre el mesénquima y el ectodermo en la mayoría de los métodos estudiados. En las tinciones realizadas se obtuvo siempre una preservación óptima tras la fijación por criosustitución (figura 1A). Las fijaciones con Bouin (figura IB), PFA (figura 1C), fijador de Zn (figura ID) o MAA (figuralE; tabla 1) mostraron resultados de peor calidad. La fijación PFA generó artefactos de ruptura del mesénquima. Sin embargo, la aplicación de la criosustitución reduce significativamente la aparición de dichos artefactos (figuras 1F, 1G y 1H). Results Cryosubstitution in methanol, combined with inclusion in wax or paraffin of low melting point and section cut to the microtome, seems to be a good alternative to fixation with paraformaldehyde (PFA), and sections to the cryostat or after inclusion in traditional paraffin. Preservation of antigenicity and tissue morphology are excluded in routine histological protocols. In a first experiment, sections of the caudal region of Danio re io embryos were shot with hematoxylin-pyrosirio. In these stages, the myotomes and the dorsal and anal fin folds are well developed. The fin folds are particularly sensitive to histological methods, usually showing ruptures between the mesenchyme and the ectoderm in most of the methods studied. In the stains performed, optimal preservation was always obtained after cryosubstitution fixation (Figure 1A). The fixations with Bouin (figure IB), PFA (figure 1C), Zn fixer (figure ID) or MAA (figuralE; table 1) showed worse quality results. The PFA fixation generated artefacts of mesenchymal rupture. However, the application of cryosubstitution significantly reduces the appearance of such artifacts (Figures 1F, 1G and 1H).
Estos métodos también se han aplicado a los tejidos del anfioxo, que presentan una histología muy difícil. Las figuras II y 1J muestran claras diferencias histológicas entre los métodos de criosustitución (figura II) y fijación con PFA (figura 1J) aplicados al anfioxo. Mientras que en las muestras fijadas con PFA se observan discontinuidades abundantes en las masas musculares y pérdida de integridad del conectivo, las muestras criosustituidas no presentan ningún defecto morfológico.  These methods have also been applied to amphioxus tissues, which have a very difficult histology. Figures II and 1J show clear histological differences between cryosubstitution methods (figure II) and fixation with PFA (figure 1J) applied to the amphioxus. While abundant discontinuities in muscle masses and loss of connective integrity are observed in samples fixed with PFA, cryosubstituted samples have no morphological defect.
La combinación entre la criosustitución y la cera o parafina de baja temperatura de fusión mejora la preservación histológica de los tejidos. Las figuras 2 A y B muestran tinciones con un anticuerpo contra mimecán del músculo liso de la pared del esófago del ratón fijados con PFA (figura 2A) o por criosustitución (figura 2B). Mientras que la sección fijada con paraformaldehidos muestra una inmuno-reactividad muy escasa, la sección obtenida tras la criosustitución muestra una inmuno-tinción muy fuerte y específica de las fibras musculares y vasos sanguíneos. Las figuras 2C y D muestran secciones transversales paralelas de aleta regenerante de 4 días post-amputación (dpa) obtenidas mediante nuestro método. En la figura 2C se observan una tinción por inmuno fluorescencia de Zns-5. Este anticuerpo marca específicamente células sintetizadoras de lepidotriquia o escleroblastos. La lepidotriquia es una estructura esquelética de la aleta que presenta marcadores tanto de hueso como de cartílago. Esto se puede demostrar mediante hibridación in situ del ARN mensajero del colágeno 10 (figura 2D). En este caso, las secciones paralelas permiten localizar ambos marcadores en las misma células usando metodologías tan diferentes. Por lo tanto, esta metodología permite la preservación de la antigenicidad de epitopos a la vez que mantiene la integridad de los ARN mensajeros tan sensibles a la degradación por AR asas. The combination between cryosubstitution and wax or low melting temperature paraffin improves histological preservation of tissues. Figures 2 A and B show stains with an antibody against mimechan of the smooth muscle of the mouse esophageal wall fixed with PFA (Figure 2A) or by cryosubstitution (Figure 2B). While the section fixed with paraformaldehydes shows very low immuno-reactivity, the section obtained after cryosubstitution shows a very strong and specific immuno-staining of muscle fibers and blood vessels. Figures 2C and D show parallel cross sections of regenerative fin of 4 days post-amputation (dpa) obtained by our method. Figure 2C shows an immuno fluorescence staining of Zns-5. This antibody specifically labels lepidotriia or scleroblast synthesizing cells. Lepidotriquia is a skeletal structure of the fin that has markers of both bone and cartilage. This can be demonstrated by in situ hybridization of collagen messenger RNA 10 (Figure 2D). In this case, the parallel sections allow both markers to be located in the same cells using such different methodologies. Therefore, this methodology allows the preservation of the antigenicity of epitopes while maintaining the integrity of messenger RNAs so sensitive to degradation by AR handles.
El uso de columnas de gravedad durante la criosustitución (columnas de criosustitución) también permite preservar la forma y posición relativa de los diversos tejidos en el interior de las muestras. Este método permite preservar el tejido de posibles plegamientos por manipulación (por ejemplo, mantener una aleta extendida; figura 3.1). Otro problema común en las muestras de tamaño pequeño son las transformaciones significativas de forma tras los métodos clásicos de criosustitución. Un ejemplo de ello son los embriones de Danio rerio, que, cuando se criosustituyen sobre una superficie, sufren deformaciones debida a la compresión de los tejidos contra el soporte. La caída de una gota de alcohol diluido en la columna de isopentano permite la sustitución homogénea por toda la superficie de la muestra evitando la deformación (figura 3.2).  The use of gravity columns during cryosubstitution (cryosubstitution columns) also allows the shape and relative position of the various tissues within the samples to be preserved. This method allows the tissue to be preserved from possible folding by manipulation (for example, keeping an extended fin; figure 3.1). Another common problem in small-sized samples is significant shape transformations following classical cryosubstitution methods. An example of this is the embryos of Danio rerio, who, when cryosubstituted on a surface, suffer deformations due to the compression of the tissues against the support. The drop of a drop of diluted alcohol in the isopentane column allows homogeneous substitution over the entire surface of the sample avoiding deformation (Figure 3.2).
El método objeto de la presente invención también ha demostrado ser muy útil para la obtención de secciones histológicas de muestras que intercalan tejidos blandos y duros. En estos casos, la separación de ambos suele ocurrir como artefacto durante los fenómenos de deshidratación-rehidratación o mientras es cortado al criostato o al microtomo. La sustitución directa y rápida del contenido hídrico durante criosustitución y la rigidez que esta cera o parafina aporta a los tejidos blandos solucionan estos problemas. Un ejemplo se representa en las muestras de cartílago, que se rodean de tejidos conectivos como la traquea, o de la extremidad de ratón durante el desarrollo (figura 4).  The method object of the present invention has also proven to be very useful for obtaining histological sections of samples that intersperse soft and hard tissues. In these cases, the separation of both usually occurs as an artifact during dehydration-rehydration phenomena or while being cut to the cryostat or microtome. The direct and rapid replacement of the water content during cryosubstitution and the rigidity that this wax or paraffin contributes to soft tissues solve these problems. An example is represented in cartilage samples, which are surrounded by connective tissues such as the trachea, or the mouse limb during development (Figure 4).
El método reivindicado es útil para analizar muestras teñidas con métodos histoquímicos, o con marcas fluorescentes tales como la fluorescencia endógena de organismos transgénicos. Las propiedades fluorescentes de los tejidos se mantienen durante todo el procesado histológico debido a la baja temperatura utilizada durante la fijación e inclusión.  The claimed method is useful for analyzing samples stained with histochemical methods, or with fluorescent labels such as endogenous fluorescence of transgenic organisms. The fluorescent properties of the tissues are maintained throughout the histological processing due to the low temperature used during fixation and inclusion.
En conclusión, el método objeto de la presente invención mejora la mayoría de los métodos histológicos actuales diseñados para microscopía óptica. Dicho método también mejora las propiedades antigénicas de tejidos embrionarios y adultos de anfioxo, Danio rerio y ratón, preservando su estructura y previniendo la degradación de ARN. Este método constituye una buena alternativa los métodos de histología clásica utilizados normalmente en estudios de embriogénesis de vertebrados frente a problemas tan comunes como la labilidad de epitopos o la intercalación de tejidos rígidos en tejidos blandos. Referencias In conclusion, the method object of the present invention improves most of the current histological methods designed for optical microscopy. This method also improves the antigenic properties of embryonic and adult tissues of amphioxus, Danio rerio and mouse, preserving their structure and preventing the degradation of RNA. This method is a good alternative to the classical histology methods normally used in embryogenesis studies of vertebrates in the face of such common problems as epitope lability or intercalation of rigid tissues in soft tissues. References
Akimenko MA, Johnson SL, Westerfield M, Ekker M (1995) Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish. Development 121(2):347-57. Akimenko MA, Johnson SL, Westerfield M, Ekker M (1995) Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish. Development 121 (2): 347-57.
Bechara IJ, Joazeiro PP, Marí-Beffa M, Becerra J, Montes GS (2000) Collagen- affecting drugs impair regeneration of teleost tail fins. J. Submicrosc. Cytol. Pathol. 32:273-80.  Bechara IJ, Joazeiro PP, Marí-Beffa M, Becerra J, Montes GS (2000) Collagen- affecting drugs impair regeneration of teleost tail fins. J. Submicrosc. Cytol Pathol 32: 273-80.
Beckstead JH (1994) A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues. J Histochem Cytochem. 42(8): 1127-1134.  Beckstead JH (1994) A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues. J Histochem Cytochem. 42 (8): 1127-1134.
Capdevila J., Vogan K.J., Tabin C.J., Izpisúa-Belmonte J.C. (2000). Mechanisms of left-rigth determination in vertebrates. Cell, 101, 9-21.  Capdevila J., Vogan K.J., Tabin C.J., Izpisúa-Belmonte J.C. (2000). Mechanisms of left-rigth determination in vertebrates. Cell, 101, 9-21.
Fernández B, Kampmann A, Pipp F, Zimmermann R, Schaper W (2003) Osteoglycin expression and localization in rabbit tissues and atherosclerotic plaques. Mol. Cell Biochem. 246:3-11.  Fernández B, Kampmann A, Pipp F, Zimmermann R, Schaper W (2003) Osteoglycin expression and localization in rabbit tissues and atherosclerotic plaques. Mol. Biochem cell. 246: 3-11.
Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, Odenthal J, van Eeden FJ (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1-36.  Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA, Odenthal J, van Eeden FJ (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1-36.
Hippe S, Düring K, Krezauler F (1989) In situ localization of a foreing protein in transgenic plant by inmunoelectron microscopy following high pressure freezing, freeze- substitution and low temperature embedding. Eur. J. Cell. Biol. 50:230-234.  Hippe S, Düring K, Krezauler F (1989) In situ localization of a foreing protein in transgenic plant by immunoelectron microscopy following high pressure freezing, freeze-substitution and low temperature embedding. Eur. J. Cell. Biol. 50: 230-234.
Marí-Beffa M, Mateos I, Palmqvist P, Becerra J (1996) Cell to cell interactions during teleosts fin regeneration. Int. J. Dev. Biol. Supp. 1 ; 179S-180S.  Marí-Beffa M, Mateos I, Palmqvist P, Becerra J (1996) Cell to cell interactions during teleosts fin regeneration. Int. J. Dev. Biol. Supp. one ; 179S-180S.
Monaghan P, Robertson D, (1990) Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry. J Microsc. 158:355- 63.  Monaghan P, Robertson D, (1990) Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry. J Microsc. 158: 355-63.
Muñoz-Chápuli R, Gallego A, Pérez-Pomares JM (1997) A Reaction-Diffusion Model can Account for the Anatomical Pattern of the Cardiac Conal Valves in Fish. J. Theor. Biol. 21 :233-40.  Muñoz-Chápuli R, Gallego A, Pérez-Pomares JM (1997) A Reaction-Diffusion Model can Account for the Anatomical Pattern of the Cardiac Conal Valves in Fish. J. Theor. Biol. 21: 233-40.
Murciano C, Fernández TD, Durán I, Maseda D, Ruiz- Sánchez J, Becerra J, Akimenko MA, Marí-Beffa M (2002) Ray-lnterray interactions during fin regeneration of Danio rerio. Dev. Biol. 252: 214-224. Quintana C, (1994) Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies. Micron. 25:63-99. Murciano C, Fernández TD, Durán I, Maseda D, Ruiz-Sánchez J, Becerra J, Akimenko MA, Marí-Beffa M (2002) Ray-lnterray interactions during fin regeneration of Danio rerio. Dev. Biol. 252: 214-224. Quintana C, (1994) Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies. Micron. 25: 63-99.
Santamaría JA, Marí-Beffa M, Becerra J (1992) Interactions of the lepidotrichial matrix components during tail fin regeneration in teleosts. Differentiation 49:143-50.  Santamaría JA, Marí-Beffa M, Becerra J (1992) Interactions of the lepidotrichial matrix components during tail fin regeneration in teleosts. Differentiation 49: 143-50.
Santos-Ruiz L, Santamaría JA, Becerra J (2001) Differential expression of FGF receptors during zebrafish fin regeneration. Int. J. Dev. Biol. 45: S131-S132.  Santos-Ruiz L, Santamaría JA, Becerra J (2001) Differential expression of FGF receptors during zebrafish fin regeneration. Int. J. Dev. Biol. 45: S131-S132.
Steedman HF (1957) Polyester wax; a new ribboning embedding médium for histology. Nature. 179: 1345.  Steedman HF (1957) Polyester wax; a new ribboning embedding medium for histology. Nature 179: 1345.
Usuda N, Ma HJ, Hanai T, Yokota S, Hashimoto T, Nagata T. (1990) Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes. J. Histochem. Cytochem. 38:617-23.  Usuda N, Ma HJ, Hanai T, Yokota S, Hashimoto T, Nagata T. (1990) Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes. J. Histochem. Cytochem 38: 617-23.
Westerfield, M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.  Westerfield, M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. Of Oregon Press, Eugene.

Claims

Reivindicaciones Claims
Método histológico optimizado para la preservación de epítopos antigénicos y de la arquitectura celular de tejidos de vertebrados caracterizado porque comprende la combinación de la fijación por criosustitución y la inclusión en una cera o parafina de baja temperatura de fusión. Optimized histological method for the preservation of antigenic epitopes and the cellular architecture of vertebrate tissues characterized in that it comprises the combination of cryosubstitution fixation and inclusion in a wax or paraffin of low melting temperature.
Método según la reivindicación anterior caracterizado porque comprende las siguientes fases o etapas:  Method according to the preceding claim characterized in that it comprises the following phases or stages:
a. Fijación por criosustitución, que comprende el uso de fijación alcohólica (criosustitución en metano 1) efectuada a bajas temperaturas, mediante el tratamiento de las muestras, adecuadamente preparadas, con isopentano y metanol;  to. Cryosubstitution fixation, which includes the use of alcoholic fixation (cryosubstitution in methane 1) carried out at low temperatures, by treating the samples, properly prepared, with isopentane and methanol;
b. Inclusión en cera o parafina de temperatura de fusión baja, que comprende la incubación de las muestras fijadas en diferentes soluciones (metanohbutanol 1 :1, butanol, butanolxera poliéster 1 : 1) a diferentes temperaturas, que van desde temperatura ambiente hasta 40 °C; y su solidificación en cera o parafina poliéster.  b. Inclusion in wax or paraffin of low melting temperature, which includes the incubation of the samples fixed in different solutions (methanobubutanol 1: 1, butanol, butanolxera polyester 1: 1) at different temperatures, ranging from room temperature to 40 ° C; and its solidification in wax or polyester paraffin.
c. Obtención de secciones; y  C. Obtaining sections; Y
d. Montaje de secciones.  d. Section assembly.
Método según la reivindicación anterior caracterizado porque la fijación por criosustitución comprende el uso de "columnas de criosustitución", formadas por dos tubos de un material resistente a bajas temperaturas, de tamaño variable, que permiten una sustitución sin contacto con objetos que evita que muestras blandas o con disposición definida se deformen por el soporte; así como de un recipiente intercambiable para el traspaso de las muestras de una columna de criosustitución a la otra.  Method according to the preceding claim characterized in that the cryosubstitution fixation comprises the use of "cryosubstitution columns", formed by two tubes of a low temperature resistant material, of variable size, which allow a contactless replacement with objects that prevents soft samples or with a defined arrangement, deform by the support; as well as an exchangeable container for the transfer of samples from one cryosubstitution column to the other.
Método según la reivindicación anterior caracterizado porque el recipiente intercambiable presenta un fondo mallado.  Method according to the preceding claim characterized in that the interchangeable container has a meshed bottom.
Método según cualquiera de las reivindicaciones 3 ó 4 caracterizado porque las "columnas de criosustitución" contienen, una de ellas, isopentano y, la otra, metanol, siendo el volumen de isopentano y de metanol preferentemente proporcional al volumen de la muestra. Method according to any of claims 3 or 4 characterized in that the "cryosubstitution columns" contain, one of them, isopentane and, the other, methanol, the volume of isopentane and methanol being preferably proportional to the volume of the sample.
6. Método según la reivindicación anterior caracterizado porque las muestras a fijar son transferidas a la columna de criosustitución que contiene isopentano enfriado a -80 °C. Method according to the preceding claim, characterized in that the samples to be fixed are transferred to the cryosubstitution column containing isopentane cooled to -80 ° C.
7. Método según la reivindiación anterior caracterizado porque la transferencia se realiza con la ayuda de un portaobjetos o equivalente en el que se ha colocado la muestra, en suspensión en metanol al 70%, o simplemente dejando caer la muestra en su interior.  7. Method according to the preceding claim characterized in that the transfer is carried out with the help of a slide or equivalent in which the sample has been placed, suspended in 70% methanol, or simply by dropping the sample inside.
8. Método según cualquiera de las reivindicaciones 6 ó 7 caracterizado porque las muestras se incuban en isopentano durante un tiempo determinado en función del tamaño y de la permeabilidad de la muestras.  Method according to any one of claims 6 or 7, characterized in that the samples are incubated in isopentane for a certain time depending on the size and permeability of the samples.
9. Método según la reivindicación anterior caracterizado porque dicha incubación se realiza en el rango de 1 a 10 minutos.  9. Method according to the preceding claim characterized in that said incubation is carried out in the range of 1 to 10 minutes.
10. Método según la reivindicación anterior caracterizado porque dicha incubación es de 2 minutos, tiempo válido para una gran variedad de muestras.  10. Method according to the preceding claim characterized in that said incubation is 2 minutes, valid time for a large variety of samples.
1 1. Método según cualquiera de las reivindicaciones 8 a 10 caracterizado porque el recipiente intercambiable con las muestras es transferido a la columna de metanol enfriado a -80 °C.  1 Method according to any of claims 8 to 10, characterized in that the exchangeable container with the samples is transferred to the methanol column cooled to -80 ° C.
12. Método según la reivindicación anterior caracterizado porque las muestras se incuban en metanol durante un tiempo determinado en función del tamaño y de la permeabilidad de la muestras.  12. Method according to the preceding claim characterized in that the samples are incubated in methanol for a certain time depending on the size and permeability of the samples.
13. Método según la reivindicación anterior caracterizado porque dicha incubación se realiza en el rango de 24 a 48 horas.  13. Method according to the preceding claim characterized in that said incubation is carried out in the range of 24 to 48 hours.
14. Método según la reivindicación anterior caracterizado porque dicha incubación es de 48 horas, tiempo válido para una gran variedad de muestras.  14. Method according to the preceding claim characterized in that said incubation is 48 hours, valid time for a wide variety of samples.
15. Método según cualquiera de las reivindicaciones anteriores caracterizado porque la inclusión en cera o parafma de temperatura de fusión baja comprende la incubación de las muestras fijadas a temperatura ambiente en una mezcla 1 : 1 de metanol y butanol durante 15 - 45 minutos, en función del tipo de muestra.  15. Method according to any one of the preceding claims characterized in that the inclusion in wax or low melting temperature paraffm comprises incubation of the samples fixed at room temperature in a 1: 1 mixture of methanol and butanol for 15-45 minutes, depending on of the type of sample.
16. Método según la reivindicación anterior caracterizado porque la incubación en la mezcla 1 :1 de metanol y butanol es de 30 minutos, tiempo apto para una gran variedad de muestras.  16. Method according to the preceding claim characterized in that the incubation in the 1: 1 mixture of methanol and butanol is 30 minutes, time suitable for a wide variety of samples.
17. Método según cualquiera de las reivindicaciones 15 ó 16 caracterizado porque las muestras, tras la incubación en la mezcla metanohbutanol 1 : 1, se incuban en butanol a temperatura ambiente durante 5 - 20 minutos, en función del tipo de muestra. 17. Method according to any of claims 15 or 16 characterized in that the samples, after incubation in the 1: 1 methanobubutanol mixture, are incubated in butanol at room temperature for 5-20 minutes, depending on the type of sample.
18. Método según la reivindicación anterior caracterizado porque la incubación en butanol a temperatura ambiente es de 10 minutos, tiempo apto para una gran variedad de muestras.  18. Method according to the preceding claim, characterized in that the incubation in butanol at room temperature is 10 minutes, time suitable for a wide variety of samples.
19. Método según cualquiera de las reivindicaciones 17 ó 18 caracterizado porque, tras la incubación en butanol a temperatura ambiente, las muestras son incubadas Incubar en butanol a 40 °C durante 10 minutos.  19. Method according to any of claims 17 or 18 characterized in that, after incubation in butanol at room temperature, the samples are incubated. Incubate in butanol at 40 ° C for 10 minutes.
20. Método según la reivindicación anterior caracterizado porque, tras la incubación en butanol a 40 °C, las muestras son incubadas en una mezcla de butanol y cera o parafina poliéster (1 :1) durante 30 minutos a 40 °C.  20. Method according to the preceding claim characterized in that, after incubation in butanol at 40 ° C, the samples are incubated in a mixture of butanol and wax or polyester paraffin (1: 1) for 30 minutes at 40 ° C.
21. Método según la reivindicación anterior caracterizado porque, tras la incubación en la mezcla de butanol :parafina poliéster 1 : 1, las muestras se incuban en cera o parafina poliéster tres veces a 40 °C durante 30 minutos cada vez.  21. Method according to the preceding claim characterized in that, after incubation in the mixture of butanol: 1: 1 polyester paraffin, the samples are incubated in wax or polyester paraffin three times at 40 ° C for 30 minutes each time.
22. Método según la reivindicación anterior caracterizado porque la inclusión en cera o parafina de temperatura de fusión baja finaliza colocando la cera o parafina poliéster sobre un molde y dejándolo, en última instancia, solidificar completamente a 4 °C. 22. Method according to the preceding claim characterized in that the inclusion in wax or paraffin of low melting temperature ends by placing the wax or polyester paraffin on a mold and ultimately allowing it to completely solidify at 4 ° C.
23. Método según cualquiera de las reivindicaciones anteriores caracterizado porque la obtención de secciones se realiza utilizando un microtomo, preferentemente en un lugar climatizado.  23. Method according to any of the preceding claims characterized in that the obtaining of sections is carried out using a microtome, preferably in a heated place.
24. Método según la reivindicación anterior caracterizado porque el montaje de las secciones obtenidas comprende el uso agua destilada estéril, preferentemente agua bidestilada estéril enfriada a 4 °C.  24. Method according to the preceding claim characterized in that the assembly of the sections obtained comprises the use of sterile distilled water, preferably sterile double-distilled water cooled to 4 ° C.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105241686A (en) * 2015-08-10 2016-01-13 河南科技大学 Preparation method of retina microscopic tissue slice of hynobiidae animals

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3234747A (en) * 1961-07-03 1966-02-15 Little Inc A Crystal forming and melting by varying applied pressure
GB1407305A (en) * 1972-02-01 1975-09-24 Krauss Maffei Ag Process for continuous freezing concentration of solutions
US3986346A (en) * 1975-04-16 1976-10-19 Jean Doat Preparation of concentrated extracts, for instance of coffee
EP0424999A1 (en) * 1989-10-23 1991-05-02 Niro Process Technology B.V. Process for producing concentrated food liquids

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE50114369D1 (en) * 2001-06-15 2008-11-13 Leica Mikrosysteme Gmbh Method and apparatus for preparing monolayers from cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3234747A (en) * 1961-07-03 1966-02-15 Little Inc A Crystal forming and melting by varying applied pressure
GB1407305A (en) * 1972-02-01 1975-09-24 Krauss Maffei Ag Process for continuous freezing concentration of solutions
US3986346A (en) * 1975-04-16 1976-10-19 Jean Doat Preparation of concentrated extracts, for instance of coffee
EP0424999A1 (en) * 1989-10-23 1991-05-02 Niro Process Technology B.V. Process for producing concentrated food liquids

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALGUACIL, M ET AL.: "DE LA FRIA FRANCONIA A LAPURA TECNOLOGIAVino of hielo and crioconcentrado: parecidos and diferencias", DIARIO "THE MUNDO", 20 February 2002 (2002-02-20), Retrieved from the Internet <URL:http://elmundovino.elmundo.es/elmundovino/noticia.html? vi_seccion=4&vs fecha=200202&vs noticia=1014229506> [retrieved on 20101203] *
OTERO, L. ET AL.: "Procesado of alimentos a alta presion and baja temperatura", ALIMENTACION-EQUIPOS AND TECNOLOGIA, October 2000 (2000-10-01), pages 153 - 158., Retrieved from the Internet <URL:http://www.alcion.es/Download/ArticulosPDF/al/gratis/07articulo.pdf> [retrieved on 20101207] *
RAVENTOS, M ET AL.: "Equipo of crioconcentracionde fluidos by placas of pelicula descendente", VI CONGRESO IBEROAMERICANO OF INGENIERIA OF ALIMENTOS,CIBIA VI, 2006, Retrieved from the Internet <URL:http://upcommons.upc.edu/e- prints/bitstream/2117/1360/1/ecuador%20crioc.pdf> [retrieved on 20101203] *
SMITH, D. ET AL.: "Freeze Concentration of FruitJuices. Food Processing for Entrepreneurs Series", NIANR NEBGUIDE, January 2006 (2006-01-01), Retrieved from the Internet <URL:http://elkhorn.unl.edu/epublic/live/g 1600/build/g 1600.pdf> *

Cited By (1)

* Cited by examiner, † Cited by third party
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CN105241686A (en) * 2015-08-10 2016-01-13 河南科技大学 Preparation method of retina microscopic tissue slice of hynobiidae animals

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