RE: Double Staining

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From:"Johnson, Jennifer(Hist)" <>
To:"'histonet'" <>
Date:Wed, 28 Apr 1999 12:19:18 -0400

Hi Cindy,

Recently, I had to perform double immuno-staining on human tissues.
Luckily, it was with one biotinylated antibody and one unconjugated, both
from different species.  Here are a few things that I found out that I hope
may be helpful to you. (They mostly pertain to chromagens, but that is
because that was where I had the most difficulty.)

I first worked out both antibody protocols individually (determined
incubation times and dilutions).

I had better luck using DAB as the first antibody's chromagen, followed by
either an alk phos or another 
peroxidase chromagen.  A lot of the other chromagens faded or were masked if
I used them first.  

It worked best for me to first try to localize the antigen I  expected to
find lesser of in the darker color (DAB!!), followed by the antigen I found
in greater quantity.  If they are both the same, I would try using the more
tricky or finicky antibody first. 

For my experiments, I found vector Red and Blue to be the best color
combinations with DAB.  Vector labs has a great web site that I got a lot of
info from.  They have charts that you can reference to see what color
combinations work together.  I would however, avoid the Nova red-- it is
kind of bricky colored and hard to distinguish between that and light DAB or
vector red.  

The web site I think is  The technical people were very
helpful too.  I used their kits and followed their protocol with just a few
tiny modifications.  

	Please contact me directly if you have any questions that I might be
able to help with or if you would like a copy of my protocols.....
	Good luck!
	Jennifer Johnson

> ----------
> From: 	Cindy Chard-Bergstrom
> Sent: 	Tuesday, April 27, 1999 7:36 AM
> To:
> Subject: 	Double Staining
> Dear Histonetters,
>     I will soon be working on a double staining IHC project. I seem 
> to remember someone discussing a proceedure that involved stripping 
> off the first primary, after addition of the chromogen, and then 
> applying the second primary. Does anyone have this protocol or any 
> others involving double staining. One project involves two rabbit 
> primary antibodies and there is not enough antibody to conjugate 
> either one to HRPO or alk-phos. Thanks to all in advance. 
> Cindy Chard-Bergstrom, BS, HT(ASCP)

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