Re: % Of Methanol Used to Dilute OOOPS

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From:Connie McManus <>
To:"J. A. Kiernan" <>, Bruce Abaloz <>
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Sorry folks... I just sent a post replying to this and for some odd reason
I didn't see METHANOL in the original post.  So, all I can say is...
Nevermind  *g*  

I don't use methanol for this purpose.

COnnie M

At 01:31 AM 06/22/2000 -0400, J. A. Kiernan wrote:
>On Fri, 23 Jun 2000, Bruce Abaloz wrote:
>> ... what % of methanol that normally used to dilute Hydrogen peroxidase 
>> for Hydrogen peroxide quenching in immunohistochemistry.
>  Assuming "hydrogen peroxide quenching" means permanently inhibiting
>  endogenous peroxidase activity in the tissue:
>    Usually a small volume of quite concentrated aqueous H2O2 is
>    added to methanol, so the concentration of methanol will be
>    over 99%. Probably it isn't critical.
>    Many people find that the methanol is not necessary and 
>    dilute the strong H2O2 with water or saline. Saline (0.9%
>    NaCl in water) may be preferable for thick frozen sections
>    of tissue that has received minimal formaldehyde fixation,
>    as is often done for research on the brain. Animal tissues
>    exposed to formaldehyde for only a few hours remain
>    osmotically responsive and are damaged by a hypotonic
>    liquid such as water (Paljarvi et al. 1979. Histochemical
>    Journal 11: 267-276. A good read). If you use H2O2 in 
>    methanol you will, of course, permanently permeabilize all
>    cell membranes and coagulate all not-yet-fixed proteins.
>    This may or may not be advantageous to your investigation.
>    Trying out is the only way to find out.
>    Always when doing immunohistochemistry you must test every
>    variation in technique, include known positive control
>    sections, and do the usual controls to exclude false positive
>    stainings. Endogenous peroxidase-like activity can be a pain,
>    as in places where many red blood cells and granulocytes are
>    present, or if you're studying certain regions of the brain
>    that contain peroxidase-positive neurons that might also
>    harbour the antigen you're looking for. 
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON,  Canada  N6A 5C1
Connie McManus
Veterinary Diagnostics Lab
Utah State University
Logan, UT

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