[Histonet] Fluorescent triple staining

[Patsy Ruegg]

If you are doing this all on the same section at the same time, I can see
that you would have trouble using a secondary that is the same species of
one of the primary antibodies.  As for blocking, I use a 5-10% solution of
non-immune serum from the host of the secondary antibodies and I would make
a cocktail of all three.  I use this block before the primary and then again
before the secondary antibody which results in very specific staining
results in my hands.  In my experience non-specific staining or background
staining is usually unwanted binding of the secondary reagent.
Patsy 


Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
P-720-859-4060
F-720-859-4110
wk email pruegg <@t> ihctech.net
web site www.ihctech.net
 

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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of James Watson
Sent: Wednesday, August 17, 2005 12:42 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Fluorescent triple staining

A coworker is staining with three primary antibodies of the following
species: rabbit, goat, and mouse.  He wants to use a goat anti-rabbit,
rabbit anti-goat, and goat anti-mouse secondary antibodies.  The questions
are, will we get cross reaction between the secondary antibodies?  What
would be the recommended blocking solution? I recommended that he use
secondary antibodies that are not of the same species of any of the
primaries, then we could cocktail the blocking serums, primary antibodies,
and secondary antibodies.  Any other suggestions?  Thank you.

James Watson HT, ASCP
Facilities Manager of Histology
GNF, Genomics Institute of the Novartis Research Foundation Room C015
858-332-4647
jwatson <@t> gnf.org 

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