[Histonet] Request for closure

[Webster, Paul]

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 <@t> lists.utsouthwestern.edu on behalf of SMITH,REBEKAH FELICIA
Sent:	Wed 6/23/2004 6:21 PM
To:	t-sherman <@t> comcast.net; histonet <@t> lists.utsouthwestern.edu
Cc:	
Subject:	Re: [Histonet] Request for closure

Todd,
 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!
SMITH,REBEKAH FELICIA
"You are a child of the universe, no less than the trees and the 
stars
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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