WO2013172796A2

WO2013172796A2 - Coverslip processor for staining of specimens on coverslips and method for staining of specimens on coverslips

Info

Publication number: WO2013172796A2
Application number: PCT/SI2013/000031
Authority: WO
Inventors: Borut JERMAN
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-05-15
Filing date: 2013-05-15
Publication date: 2013-11-21
Anticipated expiration: 2014-11-15
Also published as: SI24086A; WO2013172796A3

Description

COVERSLIP PROCESSOR FOR STAINING OF SPECIMENS ON COVER- SLIPS AND METHOD FOR STAINING OF SPECIMENS ON COVERSLIPS

The present invention relates to a coverslip processor for staining of specimens on coverslips and a method for staining of specimens on coverslips.

BACKGROUND OF THE INVENTION

The staining of biological specimens is an essential standard method used daily in clinical and research laboratories in great numbers. Many different techniques used for staining specimens mounted or attached to a glass microscopic slide or coverslip are in use in immunohistochem- istry, immunocytochemistry, in situ hybridization, in situ polymerase chain reaction and others.

Current techniques for staining biological specimens on coverslips rely on a sedentary approach where different staining fluids are placed over a biological specimen and incubated for an appropriate period of time. This technique involves the possibilities of detachment of cells or tissue, of drying of cells or tissue if the staining fluid does not cover the entire specimen, or of pooling of the stain resulting in uneven staining. To stain a biological specimen fixed on a glass coverslip, it is necessary to bring a staining liquid or reagent into even contact with the specimen on the coverslip.

For the present purposes it will be understood that cytochemistry is the technique for identification and localization of different chemical compounds, including but not limited to DNA, RNA, proteins or membranes, and their activities within the cells. Current well known techniques are immunohistochemistry, immunocytochemistry, in situ hybridization, in situ polymerase chain reaction and other. For the present purposes it will be understood that a microscopic slide is a thin, flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a light microscope, which are usually used together with a coverslip or cover glass, a smaller and thinner sheet of glass, typically 20X20X0.2 mm thick or circular, that is placed over the specimen. Specimens are usually mounted or attached to the microscope slide. The main functions of the cover slip being to keep solid specimens pressed flat, to hold the specimen in place and to protect the specimen from dust and accidental contact. It also protects the microscope's objective lens from contacting the specimen and vice-versa.

Biological specimens may be stained directly on the coverslip instead of on the microscope slide, before placing it on the microscope slide. For the present invention, the important function of the coverslip is that microbial or mammalian cell cultures or tissue culture can be grown directly on the coverslip which is placed in tissue culture plates. The use of coverslip to hold objects for examination instead of microscope slide can be an essential step when cell or tissue culture should be grown before growth structures of cell or tissue cultures are investigated under a light microscope. Each sterile coverslip is placed into sterile tissue culture plate before growth medium is added and cells or tissues are seeded and cultured. Cultured cells or tissue, after an appropriate time, attach to the coverslip.

For the present purposes it will be understood that the term staining includes application of various liquids or reagents to the specimen during staining process, even if a particular liquid does not itself produce the actual staining. These liquids or reagents usually involve the use of antibodies, enzymes, molecular probes and other expensive reagents and it is desirable to keep reagent volume used to a minimum to lower costs.

Immunocytochemistry is a technique used to analyse characteristics and structures of whole cells based on staining with various liquid reagents, commonly containing labelling dye conjugated to antibody. Live cells are seeded over a glass coverslip, placed into a sterile tissue culture plate under a sterile hood and cultured. After appropriate incubation, cells adhere on coverslip. After the incubation period, the cell or tissue culture can be treated with chemical agents or physical factors. For immunocytochemical staining procedures, cells or tissue re- quire a sequence of steps as follows: washing, fixing, permeabilizing, unmasking of cellular epitopes, blocking of background epitopes, incubation with the required antibody, secondary washing of samples, incubation with secondary antibody conjugated with labelling dye, or addition of detection compound. This is generalization of a technique that is used to analyse structures and proteins in cell or tissue culture. This method also requires transporting cover- slip from a tissue culture plate to a working microscope, in order to analyse cellular staining visually. The most frequent drawbacks of these techniques are the large volumes of liquids required or, alternatively, tissue drying and stain pooling, which could subsequently result in low reproducibility.

Current inventions address different methods and apparatus for manually staining samples on a microscope slide. An apparatus and method for efficient processing of tissue samples in slides (Pat.No. US 5958341), A method and apparatus that increases efficiency and reproducibility in immunohistochemistry and immunocytochemistry (Pat. No. US 2008/0081368) and A slide staining device (Pat. No. US 3837795, Pat. No. US2005/0179999) address all important issues when samples are processed on a microscopic slide, such as tissue drying and stain pooling, efficacy and reproducibility and re-use of some stains without loss of sensitivity. Also the labour intensive nature of staining biological specimens was overcome by automated staining methods and devices (Pat. No. US 7025937, Pat. No. US 6436348, Pat. No. US 6017495, Pat. No. US 5439649).

Methods and apparatus for handling and applying coverslips are patented or on the market, such as a processor for holding coverslips or slides during sterilization prior to their use in preparing cell cultures or for adhering or attaching cells thereto (Pat. No. WO 01/51099), a method and apparatus for detecting a microscope slide coverslip (Pat. No. WO 2010/080131), an apparatus for automatic application of cover slips on microscope slides (Pat. No. WO 94/14097, Pat. No. WO 95/20176, Pat. No. US 2003/0047863, Pat. No. US 2004/0092024), and a device for automatically attaching cover slips to microscope slides (Pat. No. US 6382693). Other inventions, such as an apparatus for handling coverslips, could overcome the associated labour intensive handling with coverslip (Pat. No. US 3972423). Bear F., An Apparatus Designed for Holding Cover Slips During Fixation and Staining, Biotechnic and His- tochemitry, 1929, Vol. 4, No. 2 , pages 59-60, describes an apparatus or holder for fixing and staining cover slips upon which cell cultures are grown. The holder described by Bear is made with grooves on the sides and on the bottom into which the cover slips fit. The holder described by Bear enables high throughput processing of cell culture samples on coverslips, but does not address still the most frequent drawbacks of these techniques, such as minimize volumes of liquids required, tissue drying and stain pooling and exposure specimens to light. Additionally Poole G. M., Apparatus for Fixing, Staining, and Rinsing of Tissue Cultures for Fluorescent- Antibody Testing, Applied Microbiology, 1972, Vol. 24, No. 2, pages 281-282, describes a staining tray and tray-housing container to facilitate fluorescent-antibody staining of tissue cultures on cover slips, which allows fixing, staining, and rinsing with a minimum of handling. Although this apparatus addresses issues such as lower misidentity and breakage due to minimal handling required, may not overcome previously presented frequent drawbacks, such as tissue drying, stain pooling and do not prevent exposure of specimens to light.

It is therefore an object of the present invention to provide a coverslip processor which overcomes the difficulties and disadvantages of the prior art, and that especially enables efficient and high throughput cytochemical staining of cell culture samples on coverslips, and provides minimal volume of chemicals or antibodies used for staining, overcomes background signals, increase reproducibility and sensitivity of sample staining and prevents exposure specimens to light.

Additionally, a method for staining utilizing such a coverslip processor shall be described.

The first object is achieved by a coverslip processor for staining of specimens on coverslips, the processor comprising a rack, and a top slider and a base seal for closing the rack, wherein the rack is partitioned with a number of compartments, each compartment being large enough to accommodate a coverslip.

The second object is achieved by a method for staining of specimens on coverslips, preferably cytochemical staining, using the coverslip processor according to the invention. Preferred embodiments are disclosed in the sub claims.

Surprisingly, it was found that the inventive coverslip processor allows high throughput cyto- chemical staining which, used with the common staining techniques, could help the scientific community to speed progress in molecular cell research. Especially, the inventive Coverslip processor and the method allow users an easy and safe handling with multiple, preferably glass, coverslips, staining of cells grown on glass coverslip in a high throughput format, which shortens time required for a preparation of staining, significantly decreasing the amount of chemicals or antibodies used for staining, overcoming background signals, increasing reproducibility and sensitivity of samples staining, since preventing exposure to light.

SUMMARY OF THE INVENTION

The present invention is a coverslip processor and a method for performing manual high throughput cytochemical staining of cells grown on glass coverslips. A single coverslip processor presented here is capable of holding preferably up to 8 specimens on glass coverslip paralleled, even can be made for airy number. Coverslips are inserted to a single processor which enables simultaneously progression through all the steps of the staining procedure from fixing, washing, staining without ever separating the coverslips from the coverslip processor. These procedures are labour intensive and the ability to process, with or without different staining agents, up to 8 coverslips at once rather than individually at consistent conditions is an important aspect of the invention. The coverslip processor comprises a rack which is secured with removable top slider and base seal. The processor with removable top slider and base seal is designed to result in minimal staining volumes required, no stain pooling, no drying of cells or tissue which are commonly encountered problems in standard sedentary immu- nocytochemical techniques. The processor provides stable transport for liquid immersed specimens attached on coverslips during the techniques used for cytochemistry. This results in better consistency and increases the sensitivity and reproducibility of the assays. The base of the rack is divided into individual compartments, each holding preferably one standard laboratory coverslip, with a biological specimen attached to the coverslip. This allows processing with the same or with different staining agents preferably up to 8 coverslips at once, where other processing conditions are the same. The sliders are of the same or similar material as the base of processor. The processor material has preferably a smooth surface, low affinity for antibodies and is stable at high temperature for autoclaving and do not emit or reflect light, which importantly increases the sensitivity and reproducibility of the assays. The processor design allows easy cleaning, since there are no dead angles or comers.

Standard laboratory coverslips with attached specimens are placed into the rack of the processor with inserted base seal. Through the top of the cavity, enough solution is placed into each compartment to cover the specimen on the coverslip. The top slider is then closed for incubation. Both, top slider and base seal are removed and the rack of the processor is placed into washing solution for specimen rinsing.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a top slider.

FIG. 2 shows a base seal.

FIG. 3 shows a top view of the rack of the coverslip processor with top slider.

FIG. 4 shows a top view of the rack base of the coverslip processor without top slider.

FIG. 5 shows a side sectional view of the rack of the coverslip processor without top slider and base seal.

FIG. 6 shows a bottom view of the rack base of the coverslip processor where the seal is off. FIG. 7 shows a cross sectional view of the rack of the coverslip processor with inserted cover- slips and with top slider and base seal.

FIG. 8 shows a cross sectional view of the rack of the coverslip processor without top slider and base seal.

FIG. 9 shows an end cross sectional view of the coverslip processor with inserted coverslip and with top slider and base seal closed at the rack.

FIG. 10 shows an end cross sectional view of coverslip processor without top slider and base seal at the rack.

FIG. 11 shows an end sectional view of coverslip processor with top slider and base seal attached at the rack.

FIG. 12 shows an end sectional view of coverslip processor without top slider and base seal attached at the rack.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a coverslip processor and a method for performing manual high throughput cytochemical staining of cells grown on glass coverslips.

The rack of the coverslip processor can be secured with removable top slider and base seal.

FIG. 1 illustrates throughout a top slider 2, top view of a top slider 2a, side sectional view of a top slider 2b and end sectional view of a top slider 2c. FIG. 2 illustrates throughout a base seal 3, top view of a base seal 3 a, side sectional view of a base seal 3b and end sectional view of a base seal 3 c. Raised portion 12 of the base seal ensures a liquid tight seal arid minimizes the required volume, when a coverslip processor 1 is bottom closed and for example staining is performed. Top slider 2 and base seal 3 are preferable from the same material as a rack 1 of the coverslip processor. Top slider 2 and base seal 3 ensure watertight closed compartments 6 to reduce evaporation and exposure of specimen to light.

FIG. 3 is an illustration of a top view of the rack 1 of the coverslip processor where top slider 2 is closed.

FIG. 4 illustrates the top view of the rack 1 of the coverslip processor. The single rack 1 presented here is preferably capable of holding Up to 8 specimens on glass coverslips 11 paralleled, evert can be made for any number. Coverslips 11 are inserted to a single rack 1 which enables simultaneously progression through all the steps of the staining procedure from fixing, washing, staining without ever separating the coverslip from the rack 1 of the coverslip processor. The body 4 of the rack 1 is preferably made of any material with nature that has a heat resistance up to 150 °C or above, has resistance to chemical agents, such a polar and nonpolar agents, strong acids or bases and resistance to corrosion. This is necessary to withstand staining procedure, autoclaving and/or microwaving processing of sliders and rack 1 which is necessary in some procedures that coverslip processor can be used in. The coverslips holder is preferable of any material that do not emits or reflect light.

A top slider 2 clip 5 is located at the top of the rack 1 at each side, to allow insertion of top slider 2. Each compartment 6 with recess 7 is preferably large enough to fit one standard coverslip 11. The compartments 6 are distant from each other enough to fit standard multichannel pipette, which allows processing up to 8 coverslips at once. Small locating shoulder 8 allows that coverslip 11 is removed with tweezers from rack 1 and to locate the pipette when liquids or reagents is added. Small 8 and large locating shoulders 9 are designed as place for efficient staining even when the cells or tissue are not as monolayer. The compartment 6 is positioned at angle, preferable 70 degrees, which allows insertion of coverslip 11 with attached cell or tissue oriented up and addition of liquids or reagents through small locating shoulder 8 at the coverslip back side to prevent suction off part or all the cells or tissue culture attached to the coverslip 11. An addition of liquids or reagents at the back side also efficiently removes unwanted cells, if present, at the back side of the coverslip, which could step into contact with objective lens after coverslip is placed on a microscope slide. Locating shoulders and compartment angle thus prevent cells or tissue detaching from the coverslips and increase efficacy of staining and washing of the specimens.

FIG. 5 illustrates a side view of the rack 1.

FIG. 6 illustrates the bottom view of the rack 1 , where the body 4 and waste liquids exit point 10 are shown.

FIG. 7 illustrates a cross sectional view of FIG. 3, with top slider 2 and base seal 3 and inserted coverslips 11. Standard laboratory coverslips 11 with attached specimens are placed into the rack 1 with inserted base seal 3. Through the top of the cavity, enough solution is placed into each compartment 6 to cover the specimen on the coverslip 11. The top slider 2 is then closed for incubation.

FIG. 8 is an illustration of a side cross sectional view of the rack 1, showing the walls 4, compartment module 6, liquids entry point at the small locating shoulder 8 and waste liquids exit point 10. Compartment module 6 is narrowed at point 13 that prevents coverslip 11 to fall through. Fig. 8 is for a situation, when top slider 2 and base seal 3 are removed and the processor is placed into washing solution for specimen rinsing.

FIG. 9 is an illustration of an end cross sectional view of coverslip processor with the rack 1 being closed with top slider 2 and base seal 3 and with coverslip 11 positioned into compartment 6. FIG. 10 is an illustration of an end cross sectional view of coverslip processor with the rack 1 being in open state without coverslip 1 1 positioned into compartment 6.

FIG. 11 is an illustration of an end sectional view of the coverslip processor where both sliders are closed at the rack 1.

FIG. 12 illustrates an end side view of the coverslip processor. The body 4 and top slider 2 clip 5 are shown.

A generalized example of the immunocytochemical method used with the coverslip processor is as follows:

1. The processor with rack 1 and open top slider 2 and base seal 3 (FIG. 4) contains a number of compartments 6, and therefore a number coverslips 11 are inserted, each into one recess 7 located in compartment 6. Coverslip 11 with attached cells or tissue is oriented up.

2. The specimens are washed by immersion of the rack 1 in washing fluid; both top slider 2 and base seal 3 are open.

3. The specimens are fixed by introducing fixing solution; base seal 3 is closed, and top slider 2 is open.

4. The specimens are washed by immersion of the rack 1 in washing fluid; both top slider 2 and base seal 3 are open.

5. The specimens are blocked with blocking solution, where base seal 3 is closed, and top slider 2 is open. 6. Solution of primary antibody is added, where base seal 3 is closed, and top slider 2 is open. As incubation starts, both the top slider 2 and the base seal 3 are closed (FIG. 7 and FIG. 9).

7. The specimens are washed by immersion of the rack 1 in washing fluid; both top slider 2 and base seal 3 are open.

8. A fluorescence conjugated secondary antibody is added, where base seal 3 is closed and top slider 2 is open. As incubation starts, both the top slider 2 and the base seal 3 are closed (FIG. 7 and FIG. 9).

9. The specimens are washed by immersion of the rack 1 in washing fluid; both top slider 2 and base seal 3 are open.

10. The coverslips 11 are removed with tweezers and placed on a microscope slide.

The features disclosed in the foregoing description, the claims and the accompanying drawings may, both separately and in any combinations thereof, be material for realizing the invention in diverse forms thereof.

Claims

1. Coverslip processor for staining of specimens on coverslips, wherein the processor comprising a rack (1), and a top slider (2) and a base seal (3) for closing the rack (1), wherein the rack (1) is partitioned with a number of compartments (6), each compartment (6) being large enough to accommodate a coverslip (11).

2. Coverslip processor according to claim 1, wherein the top slider (2) and the base seal (3) provide water tightness when connected to the rack (1).

3. Coverslip processor according to claim 1 and 2, wherein each compartment (6) has a locating shoulder (8) and a recess (7) to accommodate a coverslip (11).

4. Coverslip processor according to any of the preceding claims, wherein the rack (1), the top slider (2) and/or the base seal (3) are made of a material capable of withstanding an autoclaving process, withstanding chemical agents and/or withstanding corrosion.

5. Coverslip processor according to any of the preceding claims, wherein the rack (1), the top slider (2) and/or the base seal (3) are made of a material that does not emit or reflect light.

6. Coverslip processor according to any of the preceding claims, wherein the compartments (6) are distant from each other to fit a standard multichannel pipette.

7. Coverslip processor according to any of the preceding claims, wherein the compartments (6) are water tightly closed against each other.

8. Coverslip processor according to any of the preceding claims, wherein the compartments (6) are inclined from the vertical.

9. Method for staining of specimens on coverslips, preferably cytochemical staining, by using the coverslip processor according to any of the preceding claims.

10. Method according to claim 9, wherein the staining comprises the steps of washing, fixing, permealizating, unmasking, blocking and/or incubating.