RE: Mixing primary and secondary before staining?
It's worth a try. That is how some of the "mouse on mouse" kits work. It
would be best if you had labeled Fab fragments to work with for the
secondary because using a whole antibody may cause some steric hinderance of
primary labeling and so reduce signal. Try a few different diutions of 2nd
vs primary though.
From: Ed Boyden [mailto:firstname.lastname@example.org]
Sent: Tuesday, April 29, 2003 2:35 PM
Subject: Mixing primary and secondary before staining?
I have a large number of brain samples to process for immunocytochemistry
with a primary and then a fluorescent secondary antibody. I was wondering if
it would be possible to save steps by premixing the primary and fluorescent
secondary together, then staining with the mix and doing washes like normal.
Blocking with serum would preceded the staining, and thus should prevent any
nonspecific background. Also, since only the primary and secondary are
present when they are binding to each other, this might actually reduce
nonspecific binding to parts of the tissue.
Any ideas? I have not heard of anyone doing this before, but there were no
contraindications either listed in the histonet archives.
Dept. of Molecular and Cellular Physiology
Stanford Neuroscience Program
Stanford, CA 94305-5345
phone (650) 725-7564
fax (650) 725-8021
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