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Author Topic: Antigen Retrieval-Protease Method  (Read 4097 times)

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Offline ihcwor2

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Antigen Retrieval-Protease Method
« on: March 16, 2003, 10:16:52 PM »
Antigen Retrieval-Protease Method


Solutions and Reagents:

A.   10x Phosphate Buffered Saline (10xPBS):
Na2HPO4 --------------------- 10.9 g
NaH2PO4 --------------------- 3.2 g
NaCl ---------------------------- 90 g
Distilled water -------------- 1000 ml
Mix to dissolve and adjust pH to 7.4

B.   1x PBS:
10xPBS ---------------------- 100 ml
Distilled water --------------- 900 ml

C.   Protease Stock Solution (1% in Distilled Water):
Protease ------------------------ 100 mg
             Distilled water ------------------ 10 ml
             Mix to dissolve. Aliquot and store at -20 C

D.   Protease Working Solution (0.1% in DW):
Protease Stock Solution (1%) -------- 1 ml
Distilled water ---------------------------- 10 ml
Adjust to pH 7.8 using 1N NaOH.

E.   Protease Working Solution (0.05% in DW):
Protease Stock Solution (1%) -------- 1 ml
Distilled water --------------------------- 20 ml
Adjust to pH 7.8 using 1N NaOH.


Procedure:

1.   Deparaffinize sections in 2 changes of xylene, 10 minutes each.
2.   Hydrate in absolute alcohol, 5 minutes each.
3.   95% alcohol for 5 minutes.
4.   75% alcohol for 5 minutes.
5.   Wash the sections in distilled water.
6.   Incubate in protease working solution for 10-20 minutes at 37 C. Time of incubation may vary depending on tissue type and degree of fixation, thus optimization may be necessary.
7.   Allow the sections to cool for 20 minutes.
8.   Rinse 2x2 min in PBS.
9.   Block endogenous enzyme activities if necessary.
10.   Wash slides in PBS.
11.   Proceed to standard immunohistochemistry staining procedure.

Antigen Retrieval-Protease Method
« on: March 16, 2003, 10:16:52 PM »