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Author Topic: Antigen Retrieval-Water Bath Method (Citrate Buff  (Read 6067 times)

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Offline ihcwor2

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Antigen Retrieval-Water Bath Method (Citrate Buff
« on: March 15, 2003, 02:50:42 AM »
Antigen Retrieval-Water Bath Method (Citrate Buffer)


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.   0.01M Citrate Buffer, pH 6.0
Citrate acid (anhydrous) ----------------- 1.92 g
             Distilled water ------------------------------ 1000 ml
             Adjust pH to 6.0 with 1N NaOH.

D.   0.01M Sodium Citrate Buffer, pH 6.0
Tri-Sodium citrate ----------------------- 2.94 g
Distilled water ---------------------------- 1000 ml
Adjust pH to 6.0 with 1N HCl.


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.   Pre-heat the water bath to 95-100 C.
7.   Immerse the slides in glass staining dish containing citrate buffer or sodium citrate buffer, pH 6.0. Place the lid loosely on the staining dish, and center the dish inside the water bath and incubate the slides for 30 minutes. Turn off the water bath.
8.   Place the staining dish at room temperature and allow the slides to cool for 20 minutes.
9.   Wash slides 2x2 min in PBS.
10.   Block endogenous enzyme activities if necessary.
11.   Wash slides in PBS.
12.   Proceed to standard immunohistochemistry staining procedure.

Antigen Retrieval-Water Bath Method (Citrate Buff
« on: March 15, 2003, 02:50:42 AM »