RE: Mixing primary and secondary before staining?

From:"Morken, Tim"

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.

Tim Morken
CDC Atlanta 

-----Original Message-----
From: Ed Boyden [] 
Sent: Tuesday, April 29, 2003 2:35 PM
Subject: Mixing primary and secondary before staining?

Dear All,

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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