Re: [Histonet] Request for closure

From:Gayle Callis

There is another endogenous peroxidase blocking method the blocks
peroxidases and pseudoperoxidases.  It can be used for minimally fixed
frozen sections and in your case, paraffin sections.  The is very thorough
and gentle.  It is called Glucose Oxidase blocking method.  We love it for
frozens as it does not chew sections off the slide. We have not tried it on
paraffin but I have it by private communication it will work with paraffin
sections/NBF fixed. 

I have seen it reported as 30 min in the working solution but original
publication used it for 60 min.  

I will be happy to attach the method to you, not via Histonet. 

At 09:21 PM 6/23/2004 -0400, you wrote:
> 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
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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