Anti - C3d complement (da)

Thủ tục nhuộm IHC-P

1. Deparaffinize the section in 3 changes of xylene, 10 minutes each.
2. Wash the section in 96%, 80% and 70% benzyl alcohol for 10 minutes each.
3. Rinse in distilled water, 2 x 5 minutes.
4. Block the endogenous peroxidase by incubating the tissue in 3% hydrogen peroxide (H₂O₂) for 10 minutes.
5. Wash in distilled water, 2 x 5 minutes.
6. For antigen retrieval: immerse the slide in Tris-EDTA buffer, pH 9.0*, and incubate in water bath 40 min at 96-98˚C
7. Remove the staining to room temperature and let the slide to cool (in Tris-EDTA buffer, pH 9.0) for 20 minutes.
8. Rinse in distilled water, 2 x 5 minutes.
9. Wash in 0.05 M Tris-HCl , pH 7.6 or PBS (buffer A), 2 x 5 minutes
10. For concentrated products: Incubate the section with primary antibody diluted in buffer A at the dilution 1:100 - 200 for 1 hour in the closed wet chamber.
    For RTU products: Incubate the section with primary antibody (ready to use) for 1 hour in a closed wet chamber.
11. Wash 3 x 5 minutes with buffer A.
12. Apply the secondary antibody (the protocol depends on the supplier), and proceed to standard immunohistochemistry protocol (HRP - Peroxide - DAB).
13. Wash 3 x 5 minutes with buffer A.
14. Apply the chromogen (DAB), 10 minutes.
15. Wash in water, 2 x 5 minutes.
16. Rinse in CuSO4.5H2O solution /0,90g NaCl + 0,50g CuSO4.5 H₂O in 100ml distilled water/
17. Wash in distilled water, 1 x 2 minutes.
18. Stain in hematoxylin for 5 minutes.
19. Wash in distilled water, 3 x 2 minutes.
20. Rinse in ammonia water 37mmol/l, /2,5ml ammonium hydroxid in 1l distilled water/
21. Wash in distilled water, 1 x 2 minutes.
22. Mount the slide for observation.