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Author Topic: Antigen Retrieval-Microwave Method (Citrate Buffe  (Read 17573 times)

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Offline ihcwor2

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Antigen Retrieval-Microwave Method (Citrate Buffe
« on: March 15, 2003, 02:43:36 AM »
Antigen Retrieval-Microwave 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.   Rehydrate in absolute alcohol, 5 minutes each.
3. 95% alcohol for 5 minutes.
4. 75% alcohol for 5 minutes.
5.   Wash slides in distilled water.
6.   Immerse slides in plastic 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 microwave oven.
7.   Adjust the power level of the microwave to high. Turn on the oven for 2-5 minutes or until the solution starts to boil. Turn off the oven.
8.   Check the level of buffer in the tray and fill up with buffer if necessary to cover the slides.
9.   Repeat the steps 7 and 8 for several more times (needs to be tested for each individual antibody).
10.   Place the staining dish at room temperature and allow slides to cool for 20 minutes.
11.   Wash slides 3x2 min in PBS.
12.   Block endogenous enzyme activities if necessary.
13.   Wash slides in PBS.
14.   Proceed to standard immunohistochemistry staining procedure.

Antigen Retrieval-Microwave Method (Citrate Buffe
« on: March 15, 2003, 02:43:36 AM »