Immunohistochemistry kit experimental procedure
Instruments and equipment:
pipette, immunohistochemistry pen, microwave or pressure cooker, timer, humid chamber, staining rack, cover glass, optical microscope, bottle washer, etc.
Solution preparation:
PBS solution preparation; see the immunohistochemistry kit product manual for the preparation of EDTA tissue antigen repair solution and DAB staining solution.
Experimental temperature: 15-28°C
Experimental steps:
1. Dewaxing and hydration
1) Place the paraffin sections in fresh xylene and soak for 15 min × 2 times;
2) After removing the excess liquid, place in absolute ethanol and soak for 3 min × 2 times;
3) After removing the excess liquid, place in 95% ethanol and soak for 3 min;
4) After removing the excess liquid, place in 85% ethanol and soak for 3 min;
5) Rinse with tap water for 1 min;
6) Rinse with PBS solution for 3 min × 3 times.
2. Heat the EDTA antigen repair solution (reagent 1, antigen repair is performed using a microwave)
1) Place the deparaffinized and hydrated tissue section on the high-temperature-resistant plastic slide rack in a beaker (or repair box);
2) Add the appropriate amount of repair solution 1x EDTA antigen repair solution (solution 1, diluted 20 times with purified water) to the beaker, and the liquid surface should submerge the tissue section by a certain height;
3) Use high power heating in the microwave to bring the liquid to a boil. When it boils, switch to medium power and start the timer. The repair time is generally 20 minutes. Do not let the tissue dry during this process (there should be enough repair solution);
4) Take the beaker out of the microwave and place it in cold water to cool it down;
5) When the repair solution has dropped to room temperature, take out the slide and rinse it with PBS (pH 7.4) for 3 minutes × 3 times.
Note:
When repairing antigens, make sure that the slide is always immersed in the liquid. General situation: the amount of repair solution is 800 mL/1 rack-1500 mL/3 racks;
When removing the slides and rinsing with PBS, do not rinse against the tissue to avoid tearing it.
3. Block endogenous peroxidase
1) Dry the slides with blotting paper and use an immunohistochemistry brush to make circles around the tissue;
2) Add 100 μL of 3% hydrogen peroxide (Reagent 2) dropwise to the tissue section to block endogenous peroxidase, and incubate at room temperature for 15 min;
3) Rinse with PBS for 3 min × 3 times.
4. Block
Dry the slide with blotting paper, add normal goat serum (Reagent 3) dropwise, and seal at room temperature for 15 min to reduce non-specific staining.
5. Incubate with primary antibody
1) Dry the liquid around the tissue on the slide with blotting paper;
2) Add 100 μL of monoclonal antibody (reagent 4) dropwise, and if a negative control experiment is performed, add PBS to the tissue of the control group as a negative control, and incubate in a wet box at room temperature for 1 h or overnight at 4°C.
6. Re-warm
1) After being taken out of the refrigerator overnight at 4°C, the sample needs to be incubated at room temperature for 15 min to re-warm (this step is selected for antibody incubation overnight at 4°C, and if incubated at room temperature, proceed directly to the next step of washing);
2) Rinse with PBS for 3 min × 3 times.
7. Add Polymer Helper (Reagent 5)
1) Add Polymer Helper (Reagent 5) to the section after drying with blotting paper and incubate at room temperature or 37°C for 20 min;
2) Rinse with PBS for 3 min × 3 times.
8. Incubation with secondary antibody
1) Add Polymer-anti-mouse/rabbit IgG (Reagent 6) to the section after drying with blotting paper and incubate at room temperature or 37°C for 20 min;
2) Rinse with PBS for 3 min × 4 times.
9. Color development
1) Shake off the PBS solution and dry the section with absorbent paper;
2) Add 100 μL of freshly prepared DAB staining solution (Reagent 7: Reagent 8 = 1:49 ratio) to each section and incubate for 3-5 min. Observe the staining results under an optical microscope and do not allow the color to become too dark;
3) After color development, rinse the section with tap water to stop the color development.
10. Re-staining
Add 100 μL of hematoxylin (reagent 9) for re-staining, incubate for about 30 s–1 min, and rinse with tap water for 5 min.
Note:
Depending on the strength of the hematoxylin staining solution and the length of the incubation time, the contrast staining results in a reaction that causes the cell nuclei to appear light blue to dark blue in color.
11. Dehydration, clearing, and coverslipping
1) Place the section in 70% alcohol and soak for 2 min;
2) Place the section in 80% alcohol and soak for 2 min;
3) Place the section in 90% alcohol and soak for 2 min;
4) Place the section in 95% alcohol and soak for 2 min;
5) Place the section in absolute ethanol and soak for 2 min × 2 times;
6) Place the section in xylene and soak for 2 min × 2 times;
7) Air-dry the sections in a fume hood;
8) Add neutral gum by dropwise addition and cover the sections with a coverslip.
Judging the results:
1. The results of immunohistochemical testing must be observed and judged under a light microscope on the stained sections.
The results of immunohistochemical staining must be based on the establishment of positive tissue controls and reagent negative control tests. The interpretation of the staining results is positive (+) or negative (-).
A negative staining result is the absence of brown-yellow staining in the expected cells of the tissue.
Note: A positive control sample and a blank control test must be used in every staining process, otherwise the results cannot be used.
2. On the basis of a positive tissue control and a negative reagent control, the presence of positive staining in the examined tissue section indicates that the tissue section contains the target antigen.
3. On the basis of a positive tissue control and a negative reagent control, the absence of positive staining in the examined tissue section indicates that the target antigen is unlikely to be present in the tissue section.
4. If both the positive tissue control and the negative reagent control give a negative result, this indicates that the reagent has failed or the test was performed incorrectly, and the test should be repeated.
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