[Histonet] Request for closure

[Webster, Paul]

Hi Vinnie,

I purchased a 250ml bottle of Teleostean gelatin from cold-water fish skin (cat # G-7765) from Sigma about 6 years ago. I store it on the shelf at room temp and it is still going strong.

Blocking solutions should be chosen with care and should not contain molecules that interfere with the reaction under study.

Paul.

> ----------
> From: 	histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Vinnie Della Speranza
> Sent: 	Thursday, June 24, 2004 6:37 AM
> To: 	t-sherman <@t> comcast.net; Webster, Paul; histonet <@t> lists.utsouthwestern.edu; rockbeki <@t> ufl.edu
> Subject: 	RE: [Histonet] Request for closure
> 
> Paul,
> can you cite a source for fish skin gelatin for those of us unfamiliar with it? we use skim milk as our protein blocker with good results. one may also use powdered milk in the same manner.
> Vinnie
> 
> 
> Vinnie Della Speranza
> Manager for Anatomic Pathology Services
> Medical University of South Carolina
> 165 Ashley Avenue  Suite 309
> Charleston, SC 29425
> Ph: 843-792-6353
> fax: 843-792-8974
> 
> >>> "Webster, Paul" <PWebster <@t> hei.org> 06/24/04 12:19AM >>>
> 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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