[Histonet] non-speficic binding of secundary biotinilated antibodies

Gayle Callis gcallis <@t> montana.edu
Thu Apr 1 10:59:30 CST 2004

We avoid rabbit antibodies, this host tends to give us far more problems
with background. We always purchase our secondaries adsorbed to the host
being stained.  We use either goat antiMouse F(ab')2 frag of IgG adsorbed
to RAT diluted in 10% goat serum OR Donkey antiMouse F(ab')2 frag of IgG,
adsorbed to rat (come adsorbed to many species) from Jackson ImmunoResearch
and diluted in 10% donkey serum.    

There is a clever way to prevent binding to B cell immunoglobulins
published by e Sousa in J Exp Medicine.  We have done it when staining
mouse tissues, but it would also work when staining Rat (these critters are
rather closely related) and you might not believe it works, but the results
were very clean. I will be happy to discuss this privately. 

Sometimes you just have to add rat serum 1 - 5 % to the normal serum block,
so it would be 10% rabbit/2.5% rat, or 10% goat/2.5% rat, etc. Serum are
are heat inactivated, and normal serum blocks are applied for 30 minutes
before primary.  We prefer to dilute secondaries in the same normal serum
block, even with rat serum if that is used. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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