[Histonet] Normal serum for blocking and biotin issues

From:Gayle Callis

Because normal serums can contain endogenous biotin, we always do our
avidin/biotin block AFTER the normal serum block with very clean results.
Our normal serums are also heat inactivated at 56C for 30 -60 minutes  to
remove complement., cooled and microfiltered into sterile tubes.     

I have also heard the DAKO avidin/biotin kit is made up with stronger
concentration of avidin in order to accomodate their CSA (tyramide
amplification kit) use. 


At 09:19 PM 6/23/2004 -0700, you wrote:
>Hi Rebekah,
>I just read your final summary, having missed the original posting. It
seems that you are having a problem with background in your immunolabeling
protocols involving biotin.
>Did you know that there are many biotin-like molecules present in serum so
using serum as a blocking agent for any reaction containing biotin may
cause a problem?
>As I didn't see your original protocols I may be off track (biotinylated
antibodies perhaps?). However, knowing to avoid serum as a blocking agent
when biotin is involved may be useful to others.
>As an alternative I use 1% fish skin gelatin, a favorite of electron
microscopists who use anti-biotin antibodies or perform lectin labeling.
>Also, sodium azide is an effective inhibitor of the HRP-DAB reaction.
>Good luck.
>Paul Webster.
>Paul Webster, Ph.D.
>House Ear Institute
>2100 W. 3rd St.
>Los Angeles,
>CA 90057.
>-----Original Message-----
>From:	histonet-bounces@lists.utsouthwestern.edu on behalf of SMITH,REBEKAH
>Sent:	Wed 6/23/2004 6:21 PM
>To:	t-sherman@comcast.net; histonet@lists.utsouthwestern.edu
>Subject:	Re: [Histonet] Request for closure
> You're right. Sorry I haven't responded to everyone's suggestions 
>on my posting on blocking endogenous peroxidase in paraffin 
>sections. I think those who suggested that biotin might be the 
>problem have a point, however when I ran control sections with 
>just buffer, blocking (10% goat serum), and DAB, I still got 
>brown, even though there was no avidin for endogenous biotin to 
>bind to. Also, our lab just moved, so we can't order anything 
>until our secretary gets things sorted out. As to the antibody not 
>recognizing the sheep eNOS, I've been told that the mouse antibody 
>I'm using has been used succesfully in Western blots with sheep 
>tissue.However, I realized that my tissue has enough blood in it 
>(placenta)that that itself maybe  causing some of the brown, so 
>I've been leaving the slides in peroxide and methanol for 1 hr to 
>bleach the sections, then doing an epitope retrieval method with 
>1M Tris,then blocking, primary, secondary, conjugate,etc, then 
>using AEC as the chromogen (which thankfully is red and not brown) 
>with hematoxylin as a counterstain. I still am getting some 
>staining on controls though (both when control sections have 
>enzyme and when they have no enzyme) so if anyone can think of 
>something that I might already have around to use to block that, 
>so much the better. Otherwise, the red AEC and blued hematoxylin 
>seems to produce nice looking slides (when the tissue doesn't fall 
>off during HIER)Thank you all so much for your advice!
>"You are a child of the universe, no less than the trees and the 
>You have a right to be here and whether or not it is clear to you, 
>no doubt the universe is unfolding as it should. Therefore be at 
>peace with G-d, whatever you conceive Him to be. And whatever your 
>labors and aspirations,in the noisy confusion of life, keep peace 
>in your soul.-Max Ehrmann,"Desiderata"
>Histonet mailing list
>Histonet mailing list
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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