Re: 100 micron floating sections

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From:"Karen D. Larison" <> (by way of histonet)
To:histonet <>
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I agree with Dr. Klosen's comments:  Never use the ABC complexes in thick
sections.  We have tricks to make them work in whole mounts, but in general,
you should avoid ABC techniques.  One thing that works well in whole mounts
floating sections is PAP (peroxidase anti-peroxidase) methods, particularly if
your primary is a mouse monoclonal.  In this method, you probe for the primary
with an antibody against the species in which the antibody is raised (for
example, if your primary is a mouse, you can use unconjugated goat
and then wash, and then apply the mouse PAP (mouse anti-peroxidase that has
been precomplexed with the peroxidase enzyme).  Because the unconjugated goat
anti-mouse is bivalent, it binds both the mouse monoclonal and the mouse PAP,
thereby serving as a bridge between the reagents.  In general, direct
peroxidase conjugates aren't very stable, although Boehringer has the
reputation of being one of the better suppliers of these picky-u-nish
peroxidase reagents.  PAP methods predate ABC methods, and were specifically
developed to overcome the problem of the instability of the peroxidase direct
conjugates.  We buy the PAP reagents from Jackson Immuno Research.

Karen larison -University of Oregon

Date:          Tue, 16 Feb 1999 01:49:42 +0100
From: (Paul Klosen)
Subject:       Re: 100 micron floating sections

>Does anyone have any sure fire tips for immunostaining 100 micron floating
>sections so that the antibodies actually penetrate?  Triton,
>saponin...whatever?  Any comments or suggestions will be appreciated.
>Thanks Nancy Lemke

Try the buffered ethanol procedure.
Prepare 10%, 25% and 40% ethanol buffered with 100 mM phosphate at pH 7,4.
40% buffered ethanol needs quite some adjustments.

Take the sections up and down these ethanols for 5 to 10 min each.

I have yet to see an antibody that does no longer label after this
pretreatment. And the penetration always worked fine. According to some
authors (I borrewed their procedure, but have to look up the precise ref),
even the ultrastructure is well conserved, although not as well as if you
avoid permeabilisation. If you plan on doing preembedding EM ICC, be aware
that no procedure that allows good penetration throughout thick sections
will give you a perfect ultrastructure.

One further tip. Do not use ABC complexes !!! These are quite big and will
never well penetrate. Use either streptavidin/avidin conjugates (Perox
directly linked to streptavidin, the Boehringer reagent does wonders) or
build the complexes in situ by incubating first with the streptavidin and
then the biotinylated label. The use of streptavin conjugates also works
much better if you use Triton or Saponin permeabilisation.

If you want any further details, contact me directly.

                         (o -) O
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur - 12 rue de l'Universite
F-67000 Strasbourg, FRANCE   tel:    fax:

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