[Histonet] RE: more questions on IF
I think 1hr.h202 block of frozen is way too harsh, even with 1% compared to
3%, I have found with the T cell markers the endogenous peroxidase block is
very critical, in fact although for most of my antibodies I block after the
primary antibody, for T cell markers I have to do the peroxidase block
before the antibody. What enzyme are you using for your second cd8? There
is a great product from DAKO now called Dual Block which blocks both for
peroxidase and alk. Phos. In the same solution. It seems to be gentle
enough for frozen sections. I use it on frozen rat brains when I do cd3,
cd4, and cd8. I block for 10 min. with this BEFORE the antibody and then
use a labeled polymer detection system to avoid endogenous biotin issues.
My antibody is rat anti-mouse so I then use rab. Anti-rat secondary and then
the anti-rab. Labelled polymer with great results. I use a serum free
protein block before the primary antibody for 5 min. and then I use that
again before the secondary and before the labelled polymer, if makes for
really clean results.
Patsy Ruegg, HT(ASCP)QIHC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
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From: firstname.lastname@example.org [mailto:email@example.com]
Sent: Thursday, July 21, 2005 5:59 AM
Subject: more questions on IF
Thank you for answering my note to histonet. I have a fewe more detailed
questions if you don't mind. Do you stain with an autostainer or by hand?
Do you think it's necessary to block for an entire hour between the 2 abs.
The protocol I was given has the entire CD3 stain protocol followed by 1
hour incubation in 1% H2O2 then the second protocol for staining with CD8a.
My tissues seem to loosen starting with the hour long peroxide step. What
type of slides do you mount these tissues on? What are your thoughts?
Terri A. Prestegard
3M Center, 270-3S-05
St. Paul, MN 55144-1000
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