SI24086A - Coverslip processor for staining of specimens on coverslips and method for staining of specimens on coverslips - Google Patents

Abstract

The present invention relates to a roof slide holder for roofing slate patterns consisting of a frame of the roof slide carrier (1) and the top slide (2) and the sealing base (3) for closing the frame of the roof slide carrier (1), which is divided into a number of sections, in which each of the compartments (6) fit the roof slide; as well as the method for dyeing the samples on the roof slides using the described roof slide carrier.

Description

DESCRIPTION OF THE INVENTION

Biological sample staining is an important standard method that is widely and daily used in clinical and research laboratories. There are many different staining techniques for samples mounted or affixed to a microscope slide or slide, used in immunohistochemistry, immunocytochemistry, in situ hybridization, in situ polymerase chain reaction, etc.

Current dyeing techniques for biological samples on glass slides are based on an approach whereby different dyes are poured over a biological sample and incubated for a specified period of time. With these techniques, there is a possibility of removing cells or tissue, drying the cells or tissue when the dye does not cover the entire sample and combining the dyes, resulting in uneven staining. In order to color the sample on the slide, it is necessary to allow for uniform contact between the colorant or reagent and the sample on the slide.

In the following, the term "cytochemistry" will refer to the technique of identifying and localizing the various chemical compounds in a sample, including DNA, RNA, proteins and membranes, and determining their activities. Current well known techniques include immunohistochemistry, immunocytochemistry, in situ hybridization and in situ polymerase chain reaction and others.

Hereinafter, the term "microscopic glass" will refer to a thin, straight, translucent and usually glass piece of typical dimensions 75 mm X 25 mm, 1 mm thick, used as a carrier for examination objects under a light microscope and commonly used in conjunction with a smaller and thinner glass, transparent and usually glass piece, typically * ·

-220X20X0,2 mm, placed over the sample. Samples are usually mounted or attached to a microscope slide. The main functions of the sunroof are to maintain the strength of the specimens and the compression of the specimens in one plane, to maintain the specimens in place, and to protect the specimens from dust or unwanted physical contact. The sunroof also protects the microscope lens against contact with the specimen and vice versa.

Biological specimens can be stained on a microscope slide or directly on a slide, which is then placed on a slide. It is important for the present invention that it is possible to cultivate microbial or mammalian cell cultures or tissue cultures on a cover glass when the cover glass is placed directly in the tissue culture plate. The use of an umbrella to attach the objects to be examined under a light microscope instead of a microscope slide is crucial when cell and tissue cultures need to be grown in such a way as to form the desired cell or tissue structures to be microscoped. Each sterile glass slide should be placed in sterile plates before sterile culture medium is added and vaccinated and cultured with cells or tissues. The cultured cells or tissues attach to the slide after a certain time.

In the following, the term "dyeing" will refer to the addition of different liquids or reagents to the sample, even if the particular liquid does not in itself allow for actual coloring. These liquids or reagents may contain antibodies, enzymes, molecular probes and other expensive reagents, where it is desirable to use them in minimal quantities in order to reduce costs.

Immunocytochemistry is a technique used to analyze the properties of cellular structures based on staining with a variety of liquid reagents, usually containing an antibody conjugated to a marker dye. The live cells were grafted onto a glass slide, placed in a sterile tissue culture plate with a sterile lid, and cultured. Upon proper incubation, the cells attach to the glass slide. After incubation, the cell or tissue culture can be treated with chemical agents or physical factors. Immunocytochemical staining of cells or tissues is carried out in a sequence of steps in the following order: washing, fixing, permeabilizing, revealing cellular epitopes, blocking epitopes from the background, incubation with

-3 specific antibodies, secondary washing, incubation with secondary antibodies conjugated with the marker dye or addition of the marker substance. This is a generally described technique used to analyze structures and proteins in cell or tissue cultures. According to this method, the slide is transferred from a tissue culture plate to a working microscope where cell staining is visually analyzed. The most common disadvantages of this technique are the large volumes of liquids consumed, or the drying of the tissues and the evaporation of the dyes, resulting in a lower reproducibility.

There are inventions that relate to various methods and apparatus for manually painting samples on microscopic glass. Apparatus and method for efficient working with tissue specimens on microscopic lenses (Patent US 5958341), method and apparatus for increasing efficiency and reproducibility in immunohistochemistry and immunocytochemistry (Pat. No. US 2008/0081368) and microscopic staining apparatus ( Patent US 3837795, Patent US2005 / 0179999) ameliorate significant problems related to working with specimens on microscopic lenses, such as e.g. tissue drying, dye pooling, efficiency and repeatability, and reuse of some dyes without reducing sensitivity. Also, the technical complexity of working in the dyeing of biological preparations was circumvented by automatic dyeing methods and devices (Patent US 7025937, Patent US 6436348, Patent US 6017495, Patent US 5439649).

Methods and installations for the treatment and use of sunroofs are patented and already on the market, e.g. a carrier for holding the slide or microscope slides during sterilization before they are used to prepare or attach cells to cells (Patent WO 01/51099), method and apparatus for detecting microscopic slides (Patent WO 2010/080131), device for the automatic deposition of sunroofs on microscopic slides (Patent WO 94/14097, Patent WO 95/20176, Patent US 2003/0047863, Patent US 2004/0092024) and the device for automatically attaching sunroofs to microscopic glasses (Patent US 6382693). Other inventions such as the use of a sunroof solves the technical complexity of working with sunroofs (Patent US 3972423). Bear E, An Apparatus Designed For Holding Cover Slips During Fixation And Staining. Biotechnic and Histochemitry, 1929, Ed. 4, No. 2, on pages 59-60, he described the device or device. mounting bracket for fixing and painting the sunroof, on • ·

-4 which have grown cell cultures. The bracket described by Bear has grooves on the sides and at the bottom that fit into each deck. The carrier described by Bear provides high-performance processing of cell culture samples on glass slides, but does not solve the current and common problems such as: high consumption of fluids required for dyeing, tissue drying and mixing of dyes and exposure of light samples. Poole G. M., Apparatus for Fixing, Staining, and Rinsing of Tissue Cultures for Fluorescent-Antibody Testing. Applied Microbiology 1972, ed. 24, No. 2, at pages 281-282, described tray dyeing and a tray dyeing container that allows fixing, dyeing and rinsing with minimal handling. Although this device solves problems such as misidentification and refraction due to minimal handling, problems such as tissue drying, color pooling, and exposure to light samples are still unsolved.

The purpose of the present invention is to find and describe a holder for roof glasses that solves the problems and disadvantages of the current state, in particular, by allowing the efficient and high-performance cytochemical staining of cell culture samples on the roof glasses with minimal amounts of chemicals or antibodies consumed, reduced background signal , increased reproducibility and sensitivity of sample coloring without exposing the sample to light.

An additional purpose is to describe the method of coloring the samples pertaining to the use on this carrier of the windscreen.

The first object of the invention is achieved by a roof-glass carrier for painting patterns on the roof-glass windows, comprising a frame of a roof-glass carrier and an upper slider and a sealing base that closes the carrier, the carrier being divided into numerous sections, each section being sufficiently large, to fit one roof glass into it.

A second object of the invention is achieved by the method for staining the samples on the glass slides, in particular for cytochemical staining using the glass sling carrier, as described in the invention.

The most appropriate explanations are disclosed in the claims.

-5An surprisingly, the glass carrier supports the possibility of simultaneous cytochemical staining, which, using known staining techniques, could help the scientific community improve the advances in molecular cell research. An innovative deck holder and method for painting the samples on the roof glasses allows users to color and easily and safely handle many, especially glass, roof glasses, which shortens the time required to prepare the dye, significantly reducing the amount of chemicals or antibodies used for dyeing, it allows the background signal to be lowered, improves the reproducibility and sensitivity of the pattern coloring, and protects the samples on the skylights from light.

SUMMARY OF THE INVENTION

The subject of the invention is a carrier of the glass slides and a method for manually performing the cytochemical staining of cells grown on the slides which is carried out on several samples simultaneously. An individual deck holder is presented, capable of carrying up to 8 roof glasses with specimens in parallel, but which can be upgraded to a carrier that can carry any number of roof glasses. The sunroofs are inserted into a single carrier, allowing simultaneous passage through all the staining steps, from fixing, rinsing to painting, without having to remove the sunroof at any time from the sunroof carrier. Dyeing processes are generally labor-intensive, and the ability to treat dyes with or without dyes up to 8 sample glasses in parallel, compared to treating each sample individually under the same conditions, is an important aspect of the present invention. The sunroof carrier has a sunroof support bracket that is secured by a top slide and a sealing base. The top slider and the sealing base can be removed, and the carrier is designed to minimize the consumption of dyes and not to combine the dye, to dry cells or tissues, which are common problems common to conventional immunocytological techniques. The carrier allows stable transport into the liquid of immersed specimens affixed to the slides while using immunocytological techniques. This results in better consistency and greater sensitivity and repeatability of tests. The base of the deck holder frame is divided into separate compartments, with each compartment carrying one deck with a biological specimen attached. This allows one or different dyes to be treated with up to 8 samples at a time, with the remaining conditions being the same for all samples. The upper slider and the sealing base are made of a material similar to or the same as that used for the skylight holder.

-6 The carrier material generally has a smooth surface, low affinity for antibodies and is stable at high temperatures during autoclaving and does not transmit, emit or reflect light, significantly increasing the sensitivity and repeatability of the assays. The shape of the bracket makes it easy to clean as the bracket has no blind spots.

Standard laboratory glass slides with fixed specimens are placed in the frame of the glass slider bracket with the sealing base inserted. A sufficient amount of solution is added through the top of each cavity in the compartment to cover the sample on the slide. This is followed by incubation when the top slide is closed. Both the top slider and the sealing base are removed before washing the samples, and the carrier is immersed in the cleaning solution.

DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the top slider.

FIGURE 2 shows the sealing base.

FIGURE 3 shows a floor plan of the top-slider rack mounting frame.

FIG. 4 shows a floor plan of the baseplate holder base without the top slider. FIG.

FIG. 5 shows a side view of the skylight bracket frame without the top slider and without the sealing base.

FIG. 6 shows a bottom view of the roof rack carrier frame without a seal

-7foot.

FIG. 7 shows a longitudinal cross-section of the deck-frame carrier frame with the roof-panes inserted and with the top slider and the sealing base.

FIGURE 8 shows a longitudinal cross-section of the roof rack carrier frame without the top slider and seal.

FIG. 9 shows a cross-sectional view of the roof rack bracket with an inserted top slide and a sealing base.

FIGURE 10 shows a cross-section of a roof-glass carrier without a top slider and without a sealing base.

FIG. 11 shows an outline of a roof glass carrier with a top slider inserted and a sealing base.

FIGURE 12 shows an outline of the sunroof carrier without the top slide and without the sealing base.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a holder for a roof glass and a method for performing simultaneous manual cytochemical staining of cells grown on the glass slides.

-8 The sunroof carrier bracket is closed with a removable top slide and sealing base.

FIGURE 1 shows a complete view of the top slider 2, the top plan view of the top slider 2a, the side lynx of the top slider 2b and the outline of the top slider 2c.

FIG. 2 shows a complete view of the sealing base 3, the floor plan of the sealing base 3a, the lateral drawing of the sealing base 3b, and an outline of the sealing base 3c. The raised part 12 of the sealing base 3 allows for impermeable sealing to the liquids and minimizes the volume required when the base of the sunroof carrier 1 at the bottom is closed, e.g. while performing dyeing. The upper slider 2 and the sealing base 3 are preferably of the same material as the frame of the roof glass carrier 1. The upper slider 2 and the sealing base 3 allow watertight sealed sections 6 to reduce evaporation and exposure of light samples.

FIG. 3 is an illustration of a floor plan of the roof rack carrier 1 with the closed top slider 2.

FIG. 4 shows the floor plan of the roof rack carrier 1. The single frame of the roof rack carrier 1 preferably allows up to 8 samples to be carried in parallel on the glass roof rails 11, but the roof rack carrier frame 1 may be made for any number of patterns on the roof rails. The sunroofs 11 are inserted into the frame of the sunroof holder 1, which allows simultaneous transition between the steps of the painting process from fixing, flushing, painting, without the need to separate the sunroofs from the sunroof bracket L at any time. it is preferably made of any material that is resistant to temperatures up to 150 ° C or more, is resistant to chemicals such as flames and non-polar solvents, strong acids or bases and is resistant to corrosion. This is necessary to ensure that the sunroof carrier can be used during the dyeing, autoclaving and irradiation processes of the sunroof and the sunroof frame

-9 glasses 1 with microwaves, which is required for some uses of the sunroof carrier. The roof rack carrier frame 1 is preferably of any material which does not emit or reflect light.

The rails 5 of the top slider 2 are located at the top of the deck holder bracket 1 on each side, which allows the top slider 2 to be inserted. Each section 6 has a slot 7 that is large enough to fit one standard-sized deck. The distances between sections 6 are equal to the distances between the individual channels of the multichannel pipette, allowing simultaneous processing of up to 8 glass slides. The individual slot 7 of each section 6 has a smaller recess 8 that allows removal of the cover glass 11 from the frame of the cover glass carrier 1 with tweezers and allows the attachment of multichannel pipettes for adding liquids or reagents. The smaller recesses 8 and the larger recesses 9 are designed to allow effective staining, even if the cells or tissue are not in a single layer. The individual compartment 6 is angled, preferably 70 degrees, allowing insertion of a cover glass 11 with attached cells or tissue on the front and adding liquids or reagents through smaller recesses 8 at the rear of the cover, preventing partial or complete detachment of the part or all cells on the slide. In addition, adding reagents on the back of the slide effectively removes unwanted cells, if present on the back of the slide, from which they could come into contact with the lens of the microscope lens after the slide is placed on a microscope slide. The recesses and the angle of the sections 6 therefore prevent the cells or tissues from detaching themselves from the slide and consequently increase the staining and washing efficiency of the specimens.

FIG. 5 shows a side view of the roof rack carrier frame 1.

FIG. 6 shows a bottom view of the deck support frame 1, showing a wall 4 and an outlet for waste liquids 10.

-10FIGURE 7 shows a longitudinal cross-section of FIGURE 3, with the top slider 2 inserted, the sealing base 3 and the slides inserted 11. The standard laboratory slides or attached specimens are placed in the frame of the slider glass holder 1 with the sealing base 3 inserted. of sections 6, a sufficient amount of liquid is added from the top to cover each sample on an individual slide 11. For incubation, close the slide holder with the top slide 2.

FIG. 8 shows a longitudinal cross-section of the frame of the roof carrier 1, showing the walls 4, the section module 6, the entry point for liquids at the site of the smaller recess 8 and the exit point for waste liquids 10. The section module 6 narrows to a point 13, which prevents the top cover 11 would fall through the frame of the top cover carrier 1. FIGURE 8 refers to the condition that the top slider 2 and the sealing base 3 are removed and the carrier is placed in the sample rinse solution.

FIG. 9 shows a cross-sectional view of the sunroof carrier, where the housing of the sunroof carrier 1 is closed with the upper slider 2 and the sealing base 3 and the sunroof 11 is inserted into section 6.

FIGURE 10 shows a cross-section of a roof-glass carrier with an open frame of a roof-glass carrier 1 without a roof-glass 11 inserted in section 6.

FIG. 11 shows an outline of the sunroof bracket, wherein the skirting of the sunroof bracket 1 is closed with the upper slider 2 and the sealing base 3.

FIGURE 12 shows an outline of a roof rack holder with walls 4 and rails 5 of the top

- 11 sliders 2.

A general example of the use of immunocytochemical methods with a carrier glass is as follows:

1. The frame of the sunroof carrier 1 with the open top slider 2 and the sealing base 3 (FIGURE 4) contains a number of sections 6 and, consequently, a number of sunroofs 11 which are inserted into each slot 7 located in a single section 6. The surface of the sunroofs with attached cells but the tissue was placed vertically.

2. The samples are washed by immersing the deck holder carrier 1 in the cleaning fluid; the upper slider 2 and the sealing base 3 are open.

3. Samples shall be fixed with fixation solution; the sealing base 3 is closed and the upper slider 2 is open.

4. The samples shall be washed by immersing the deck glass carrier frame 1 in the cleaning fluid; the upper slider 2 and the sealing base 3 are open.

5. The samples are blocked with a blocking solution, with the sealing base 3 closed and the top slider 2 open.

6. A solution of primary antibodies is added, with the sealing base 3 closed and the upper slider 2 open. After incubation has started, the top slide 2 and the sealing base 3 are closed (FIG. 7 and FIG. 9).

7. The samples are washed by immersing the deck holder carrier 1 in the cleaning fluid; the upper slider 2 and the sealing base 3 are open.

8. A solution of secondary antibodies conjugated with fluorescent dye is added, with the sealing base 3 closed and the upper slider 2 open. After incubation has started, the top slide 2 and the sealing base 3 are closed (FIG. 7 and FIG. 9).

·

- 129. The samples are washed by immersing the deck holder carrier 1 in the cleaning fluid; the upper slider 2 and the sealing base 3 are open.

10. Remove the slide 11 with tweezers and place on a microscope slide.

The disclosed features of the foregoing description, the claims and the accompanying drawings may be, independently and in combinations, a material for the realization of the present invention in various forms.

Claims (9)

Claims: A roof-glass carrier for painting patterns on roof-glass windows, which consists of a frame for the roof-glass carrier (1), a top slide (2) and a sealing base (3) for closing the frame of the roof-glass carrier (1), the carrier frame However, the roof windows (1) are divided into numerous sections and each section (6) is large enough to accommodate the roof window (11). Roof glass carrier according to claim 1, characterized in that the upper slider (2) and the sealing base (3) allow watertightness when attached to the frame of the roof glass carrier (1). Roof holder according to claims 1 and 2, characterized in that each compartment (6) has a recess (8) and a slot (7) into which the sunroof (11) fits. Roof holder according to any one of the preceding claims, characterized in that the roof raft carrier frame (1), the upper slider (2) and / or the sealing base (3) are made of material that undergoes autoclaving processes. chemical substances and / or withstand corrosion. Roof glass carrier according to any one of the preceding claims, characterized in that the frame of the roof glass carrier (1), the upper slider (2) and / or the sealing base (3) is made of a material which does not emit or reflect light . Roof holder according to any one of the preceding claims, characterized in that the distances between the sections (6) are equal to the distances between the individual channels of the multichannel pipette. Roof glass holder according to any one of the preceding claims, characterized in that the sections (6) are separated from each other and sealed. -148. A sunroof support according to any one of the preceding claims, characterized in that the sections (6) are inclined with respect to the vertical. 9. A method for staining the samples on the slides, preferably cytochemical staining, using the slurry carrier according to any one of the preceding claims. The method of claim 9, wherein the staining comprises the steps of washing, fixing, permeabilizing, epitope disclosure, blocking and / or incubation.