Re: Thanks re:endogenous peroxidase, a serum blocking question
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From: | Tim Morken <timcdc@hotmail.com> |
To: | HistoNet@Pathology.swmed.edu |
Reply-To: | |
Date: | Tue, 06 Apr 1999 11:38:26 -0400 (EDT) |
Content-Type: | text/plain |
Bruce,
We use a 20% sheep serum (in tris-saline-tween20 buffer) before the
primary. We dilute the primary in the same serum-buffer, so it 'keeps on
blocking.' We don't rinse the blocking serum before putting on the
primary, only an air-blow (using the DAKO auto-stainer). We then rinse
the with the tris-saline buffer without the serum.
If you have a lot of background problems then a second serum blocker may
be necessary before applying the secondary antibody. In general, I would
rather keep the procedure simple and use as few steps as possible.
Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA
email: tim9@cdc.gov
timcdc@hotmail.com
FAX: (404)639-3043
----Original Message Follows----
From: Bruce A Rasmussen <brasmuss@osf1.gmu.edu>
To: HistoNet@Pathology.swmed.edu
Subject: Thanks re:endogenous peroxidase, a serum blocking question
Date: Tue, 06 Apr 1999 10:44:30 -0400 (EDT)
My thanks to all who wrote back with detailed responses to my endogenous
peroxidase question. If I could trouble folk with just one more
question.
I have a protocol that calls for a 5% serum block (30 min to the
secondary
animal) before and primary and before the secondary. There is much
debate
about whether the second block is necessay, if it will reduce the
signal,
or if rinsing after the serum blocks is good or bad. Some folk also
suggest that a couple of percent BSA is a good idea.
Thanks again,
Bruce
--------------------------------------
Bruce Rasmussen
Predoc Fellow in Experimental Neuropsychology
The Krasnow Institute for Advanced Study
George Mason University
Mail Stop 2A1
Fairfax, VA 22030-4444
Office:703-993-4358
Lab:703-993-4369
Home:703-765-4570
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