[Histonet] RE: BSA in immunhistochemistry

[Della Speranza, Vinnie]

You don't mention if you are doing a biotin block. Human brain has lots of biotin. Rat brain may also but I don't work with animal tissues however I'm certain others here can advise you on that issue. As I recall, the ABC kits do contain a protein block but do not contain a biotin block. I'm guessing this is what is creating the background you describe however don't underestimate the importance antibody dilution plays. If you know the protein concentration of your primary antibody, start with a dilution that yields 5-10 micrograms of protein as a dilution starting point and fine tune from there.

Vinnie Della Speranza
Manager for Anatomic Pathology Services
165 Ashley Avenue  Suite 309
Charleston, South Carolina 29425
Tel: (843) 792-6353
Fax: (843) 792-8974


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Caroline Bass
Sent: Thursday, July 31, 2008 2:15 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] BSA in immunhistochemistry

Hey Guys,

I am trying to set up EGFP IHC in rat brain and just ran the first set of
tissue through.  We just guessed at the primary and secondary antibody
concentrations, and we used an ABC kit with vector-sg substrate.  The
sections developed very quickly with lots of background.  My guess is to
decrease the primary concentration and maybe block longer.  However, we¹ve
encountered a lot of IHC protocols that use BSA during blocking.  I¹m
hesitant due to the expense though.  Any suggestions as to whether this will
help and if so is there a cheap source of BSA that I can use?

Thanks,

Caroline Bass
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