Re: % Of Methanol Used to Dilute....

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From:"J. A. Kiernan" <>
To:Bruce Abaloz <>
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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


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