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Author Topic: Antigen Retrieval-Steamer Method (EDTA)  (Read 4527 times)

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Offline ihcwor2

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Antigen Retrieval-Steamer Method (EDTA)
« on: March 15, 2003, 02:48:04 AM »
Antigen Retrieval-Steamer Method (EDTA)


Solutions and Reagents:

A.   10x Phosphate Buffered Saline (10xPBS):
Na2HPO4 --------------------- 10.9 g
NaH2PO4 --------------------- 3.2 g
NaCl --------------------------- 90.0 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.   1.0 mM EDTA, pH 8.0
EDTA  ----------------------------------- 0.37 g (Sigma Cat# E-5134)
             Distilled water ------------------------- 1000 ml
             Adjust pH to 8.0 with 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. 70% alcohol for 5 minutes
5.   Wash slides in distilled water.
6.   Immerse the slides in glass staining dish containing 1mM EDTA, pH 8.0. Place the lid loosely on the staining dish, and center the dish inside the steamer.
7.   Turn on the steamer and start the timer when the water comes to boil.
8.   Steam the slides for 20 minutes and turn off the steamer.
9.   Place the staining dish at room temperature and allow the slides to cool for 20 minutes.
10.   Wash slides 2x2 min in PBS.
11.   Block endogenous enzyme activities if necessary.
12.   Wash slides in PBS.
13.   Proceed to standard immunohistochemistry staining procedure.

Antigen Retrieval-Steamer Method (EDTA)
« on: March 15, 2003, 02:48:04 AM »