Steps of paraffin section immunohistochemistry
Dewaxing and hydration
1. successively immerse the section in xylene I for 10 min and xylene II for 10 min.
2. re-immerse in absolute ethanol I for 5 min – absolute ethanol II for 5 min – 95% alcohol for 5 min – 80% alcohol for 5 min – 70% alcohol for 5 min, then rinse twice with deionised water for 2 min.
Antigen repair (optional)
3. Place the tissue section in a repair box and add an appropriate amount of 0.01M citrate buffer (pH 6.0) or EDTA repair solution (pH 9.0) to submerge the tissue.
4. Fix in the microwave for 10 minutes on medium power (start timing when the liquid starts boiling). Do not let the tissue dry during this process.
5. Take the fixative box out of the microwave and let it cool down naturally. When the fixative solution has cooled down to room temperature, take out the slides and rinse with PBS (pH 7.4) for 3 times for 3 minutes each time (do not rinse the tissue during the rinsing process to avoid breaking the tissue).
Inactivation
6. Add the prepared 3% hydrogen peroxide (dilute 30% hydrogen peroxide in deionized water) dropwise to the sliced tissue to block endogenous peroxidase, incubate at room temperature for 15 min, rinse with PBS three times for 3 min each time.
Antibody incubation
7. Blot dry the PBS with blotting paper, add 5% normal serum (same or similar species as the secondary antibody) dropwise to the slide, and seal at 37°C for 30 min.
8. Wipe the liquid around the tissue on the slide with blotting paper, draw circles around the tissue with an oil-based pen, and then add the diluted primary antibody dropwise. If a negative control experiment is performed, add PBS to the tissue in the control group. Incubate overnight at 4 °C in a humid box after adding the primary antibody. (The optimal dilution ratio of the antibody should be determined in advance through experiments, and the time should be controlled for more than 15 hours.)
9. Rinse the sections with PBS three times for 3 min each, blot dry with absorbent paper, and then add the horseradish peroxidase-conjugated secondary antibody and incubate at 37 °C for 30 min.
Signal detection
10. Rinse the section 4 times in PBS for 3 min each time, shake off the PBS liquid, dry the section with absorbent paper, add freshly prepared DAB staining solution to each section, observe under a microscope, positive signals are brownish yellow or brown, time should be controlled well, do not allow the color to become too dark. Rinse the section with tap water to stop the color development.
11. Harris hematoxylin counterstaining, generally about 30 s-1 min, wash with water, differentiate with 1% hydrochloric acid alcohol, and then rinse with tap water to remove the blue color. (optional for staining nuclei)
Dehydrate, fix and seal the section
12. After rinsing the section in water, place it in the following order: 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, absolute ethanol I, absolute ethanol II, xylene I, xylene II. Dehydrate the section in each reagent for 2 minutes, and finally air-dry the section in a fume hood.
13. Drop neutral gum next to the tissue and cover with a cover slip. First lay one side flat, then gently lower the other side to avoid creating air bubbles. The sealed section is then laid flat in a fume cupboard to dry.
14. The dried section can be observed under a microscope or an image can be captured.
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