Re: Rabbit serum background
Re: Rabbit serum background
I was able to eliminate high background by not using BSA at all and by using a .1% triton X100 in PBS for washes only and a .2% triton X100 in PBS with 2% normal serum as my blocking solution to open the cells. I make my AB in PBS only. I use normal serum only in the blocking solution.
When I first started doing this IHC for my new boss, she had me doing 10 washes and I still had high background. I wanted to scream. I called for tech support (Chemicon) and got this good advice. It worked the first time I tried it and I use this method routinely now. I have not had background since then with any AB that I use, which is mostly rabbit and sheep. I only do 2 or 3 five min. washes. It is wonderful!
Hope it will work for you!
Kathleen Spencer H.T., LCM Supervisor
University of Tennessee
901.448.3720
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From: Kimberly L Merriam <kmerriam@tktx.com>
To: "Patterson Noelle (NIDDK)" <NoelleP@intra.niddk.nih.gov>
Cc: "Histonet (E-mail)" <histonet@pathology.swmed.edu>
Subject: Re: Rabbit serum background
Date: Wed, Apr 3, 2002, 9:01 AM
"Patterson, Noelle (NIDDK)" wrote:
> Hi.
> Do people notice a high background staining when using rabbit-derived
> antibodies and/or rabbit polyclonal serum? If so, what do you do about it?
>
> Noelle Patterson
> Biologist
> NIDDK/Navy-TAB
> Bethesda, MD 20889-5603
YES! We get terrible background when we use rabbit antibodies, we avoid them,
if at all possible. My best advise would be to try blocking with several
different things (BSA, serum, commercially available blocking agents) and play
with the concentrations of your blocking agents (such as increasing serum
concentration, etc.). Also you may want to try a combination of serums in the
block (this is very useful when staining animal tissues). Pick the blocking
reagent that gives you the least amount of background.
Kim Merriam
TKT
Cambridge, MA
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