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From: 	Abizar Lakdawalla[SMTP:abizarl@innogenex.com]
Sent: 	Wednesday, April 28, 1999 12:35 PM
To: 	mogdie@innogenex.com
Subject: 	Double Staining (can you post this to histonet)

We use the following procedure routinely in our labs. works well.

Protocol Summary for paraffin embedded sections

1.	EZ-DeWaxo Solution (removes wax and rehydrates sections simultaneously)
(3 min)
	Rinse with water or 70% EtOH

2.	EZ-DeWaxo Solution (3 min)
	Rinse with water or70% EtOH
	Wash in water (5 min)

3.	Peroxide Block, if needed (5 min)
	Wash in water (5 min)

4.	Power Blocko Reagent (a kappa-casein based blocking reagent) (3 min)

5.	Primary Antibody to 1st Antigen, 30 min RT (or as required)
	Rinse in PBS/Tween, wash (5 min), rinse again

6.	Secondary Antibody (20 min RT, 5min 37C)
	Rinse, wash (5 min), rinse in PBS/Tween

7.	Enzyme-Streptavidin (20 min)
	Rinse, wash (5 min), rinse in PBS/Tween

8.	1st Substrate* (>>10 min)
	Wash in water (5 min)

9.	Microwave in Tris-Urea (10-30 sec)
Cool (5 min), wash in water (1 min)

10.	Power Blocko Reagent (3 min)

11.	Primary Antibody to 2nd Antigen
	Rinse, wash (5 min), rinse in PBS/Tween

12.	Secondary Antibody (20 min)
	Rinse, wash (5 min), rinse in PBS/Tween

13.	Enzyme-Streptavidin (20 min)
	Rinse, wash (5 min), rinse in PBS/Tween

14.	2nd Substrate* (>>10 min)
	Wash in water

15.	Counterstain and Mount

If using HRP as the enzyme, the first substrate should be nickel-DAB (dark
brown/black), and the second substrate can be AEC (brick red). For
Phosphatase, the first substrate should be BCIP/NBT (blue/purple) and the
second can be Fast Red (bright red).

Matt Ogdie
www.innogenex.com