Re: % Of Methanol Used to Dilute OOOPS
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From: | Connie McManus <conmac@cc.usu.edu> |
To: | "J. A. Kiernan" <jkiernan@julian.uwo.ca>, Bruce Abaloz <b.abaloz@zoology.unimelb.edu.au> |
Reply-To: | |
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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
USA
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