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Author Topic: Antigen Retrieval-Pepsin Method  (Read 5551 times)

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Offline ihcwor2

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Antigen Retrieval-Pepsin Method
« on: March 16, 2003, 10:14:16 PM »
Antigen Retrieval-Pepsin 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.   Pepsin Stock Solution (1% in 10mM HCl):
Pepsin ------------------------------ 100 mg
             10mM HCl (pH 2.0) ------------- 10 ml
             Mix to dissolve. Aliquot and store at 20 C

D.   Pepsin Working Solution (0.5% in DW):
Pepsin Stock Solution (1%) -------- 1 ml
10mM HCl (pH 2.0) ------------------- 1 ml


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.   70% alcohol for 5 minutes.
5.   Wash the sections in distilled water.
6.   Incubate in pepsin working solution for 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-Pepsin Method
« on: March 16, 2003, 10:14:16 PM »