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Lab Standard Procedure for Immunohistochemistry Double Labeling
Materials
Literature
Follow Standard procedure for single antibody staining
through DAB enhancer (Step 16).
17. Rinse tissue well with distilled water and place in Tris buffer.
18. Reblock tissue with avidin/biotin block (Vector) for 15 min.
each with a buffer rinse between and after.
19. Reblock tissue with Protein Block (Dako X0909) for 10 min.
20. Drip protein block off tissue and apply 2nd primary for overnight
incubation.
21. Place slides in Tris with Tween and rinse once.
22. Wipe around tissue and apply secondary antibody for 30 min.
incubation at RT. (Dako, LSAB+ Kit, K0690)
23. Rinse with buffer.
24. Apply label (Streptavadin, Dako , LSAB+ Kit K0690) for 30 min.
at RT
25. Rinse several times with distilled water.
26. Develop second primary antibody using VIP Substrate kit (Vector
SK-4600). This is a violet chromogen. 15 min.
27. Rinse slides with distilled water several times.
28. Stain slides with Methyl Green (Vector H-3402) for 3 min. at
60C.(I preheat the Methyl Green in a water bath)
29. Rinse with distilled water, dehydrate, to xylene and coverslip
with synthetic mounting media.
Last
GLP modification 1/12/04:
Jennifer
Casati
James R. Turk
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