[Histonet] Normal serum for blocking and biotin issues
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:
>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.
>Paul Webster, Ph.D.
>House Ear Institute
>2100 W. 3rd St.
>From: firstname.lastname@example.org on behalf of SMITH,REBEKAH
>Sent: Wed 6/23/2004 6:21 PM
>To: email@example.com; firstname.lastname@example.org
>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"
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Veterinary Molecular Biology
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