RE: Double Staining
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From: | "Johnson, Jennifer(Hist)" <jjohnson3@genzyme.com> |
To: | "'histonet'" <histonet@Pathology.swmed.edu> |
Reply-To: | |
Date: | Wed, 28 Apr 1999 12:19:18 -0400 |
Content-Type: | |
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 www.vectorlabs.com. 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
jjohnson3@genzyme.com
> ----------
> From: Cindy Chard-Bergstrom
> Sent: Tuesday, April 27, 1999 7:36 AM
> To: Histonet@Pathology.swmed.edu
> 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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