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Author Topic: Antigen Retrieval-Pronase Method  (Read 6026 times)

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Offline ihcwor2

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Antigen Retrieval-Pronase Method
« on: March 15, 2003, 02:45:52 AM »
Antigen Retrieval-Pronase 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.   Pronase Stock Solution (1% pronase in PBS):
Pronase ------------------------- 100 mg
             PBS ------------------------------- 10 ml
             Mix to dissolve. Aliquot and store at – 20 C

D.   Pronase Working Solution (0.1% in PBS):
Pronase Stock Solution (1%) --------- 1 ml
PBS ----------------------------------------- 10 ml
This solution can be used for overfixed tissue and/or thicker sections. The pronase concentration should be adjusted for each individual experiment.

E.   Pronase Working Solution (0.05% in PBS):
Pronase Stock Solution (1%) -------- 1 ml
PBS ---------------------------------------- 20 ml
This solution can be used for poorly fixed tissue and/or thinner sections and the pronase concentration should be adjusted for each individual experiment.


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 pronase 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-Pronase Method
« on: March 15, 2003, 02:45:52 AM »