high bg fluoresc micrscpy

Bruce Freimark bfreimark at nexellinc.com
Tue Sep 26 16:58:26 EST 2000


I've tried absorbing antibody with tissue to some success. I your case it
would be lymph node homogenate using protease inhibitors. Absorb 2-3x, spin
out the debri and titer appropriately.

-----Original Message-----
From: Ronald Rabin [mailto:RRABIN at niaid.nih.gov]
Sent: Monday, September 25, 2000 5:00 PM
To: cyto-inbox
Subject: high bg fluoresc micrscpy



I am staining human secondary lymphoid tissues (frozen sections) with goat
and rabbit polyclonal antibodies.  The secondary antibodies are rabbit and
goat HRP respectively.  I use the Vector ABC system to amplify.

I am having huge problems with background non-specific staining.  I have
tried airfuge prep of antibodies, blocking with the serum from the same
species as the secondary antibody.  It is not the ABC system per se, or even
the secondary antibody, as signal is low without primary controls.  Anybody
know how to block nonspecific binding?

Ronald L. Rabin, M.D.
Research Fellow
Cytokine Biology Unit
Laboratory of Clinical Investigation
NIAID/NIH
Building 10/Room 11N228
10 Center Drive MSC 1888
Bethesda, MD   20892-1888
Phone:  301-402-4910
FAX:      301-402-0627



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