Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
  • G01N1/31—Apparatus therefor
  • G01N1/312—Apparatus therefor for samples mounted on planar substrates
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L9/00—Supporting devices; Holding devices
  • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0822—Slides

Definitions

• the present invention relates to a coverslip processor for staining of specimens on coverslips and a method for staining of specimens on coverslips.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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:
• the processor with rack 1 and open top slider 2 and base seal 3 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.
• 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).
• the coverslips 11 are removed with tweezers and placed on a microscope slide.

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• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Physics & Mathematics (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Analytical Chemistry (AREA)
• Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Clinical Laboratory Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Sampling And Sample Adjustment (AREA)
• Automatic Analysis And Handling Materials Therefor (AREA)

Applications Claiming Priority (2)

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• 2012
  • 2012-05-15 SI SI201200148A patent/SI24086A/sl not_active IP Right Cessation
• 2013
  • 2013-05-15 WO PCT/SI2013/000031 patent/WO2013172796A2/fr not_active Ceased

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