[Histonet] Thick section penetration for IHC

[MKing]

Bob,
There's always fluorescence--these authors used 100 um Vibratome 
sections and found that the primary antibody may make a difference too.

Journal of Neuroscience Methods 170 (2008) 165–178
Associative image analysis: A method for automated quantification
of 3D multi-parameter images of brain tissue
Christopher S. Bjornsson, Gang Lin, Yousef Al-Kofahi, Arunachalam 
Narayanaswamy, Karen L. Smith, William Shain, Badrinath Roysamb

Simple avidinylated HRP may be smaller than ABC complexes and penetrate 
better.  People have also used DMSO 1-10% in incubation solutions, and 
ethanol extraction steps on cut thick sections, reported to improve 
penetration (both in histonet archives).

Good histo,
Mike King
UF Pharmacology & Therapeutics

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Message: 3
Date: Tue, 27 May 2008 10:04:52 -0800
From: "Bob Nienhuis" <bob.nienhuis <@t> gmail.com>
Subject: [Histonet] Thick section penetration for IHC
To: Histonet <histonet <@t> lists.utsouthwestern.edu>

It has been suggested that the ABC detection system may not be the 
optimal choice for IHC in thick brain sections (~100 microns). 
Apparently, the large avidin-biotin complexes may not penetrate tissue 
too well.

What would you suggest instead, LSAB, polymer or...?

Bob Nienhuis
Neurobiology Research
UCLA / VA Medical Center
Los Angeles
-------------- next part --------------

Bjornsson CS, Lin G, Al-Kofahi Y, Narayanaswamy A, Smith KL, Shain W, Roysam B.
Associative image analysis: A method for automated quantification of 3D multi-parameter images of brain tissue.
J Neurosci Methods. 2008 May 15;170(1):165-78. 
use 100 um sections, get complete penetration with some Abs, not GFAP, 

------------------
Date: 15 Feb 1999 18:30:55 -0600
From: larisonk <@t> UONEURO.uoregon.edu
Subject: Re: 100 micron floating sections

Nancy,

When we stain whole mount embryos, we find that adding 1% DMSO to all
incubation and wash buffers enhances staining.  In fact, with the older
embryos, pre-incubating them overnight in buffer containing 10% DMSO seems to
help a great deal.  Wash thourougly before adding your primary, however.  1%
DMSO doesn't seem to affect antibody binding, whereas higher concentrations
apparently does.  In most cases, we also find that immersing the tissue
briefly
(7 minutes) in ice-cold (-20C) acetone before the blocking step also helps.
We
also use triton and tween.  The detergents, however, are harder on the tissue
than DMSO.  Tween may be a bit gentler, and with some antibodies appears to be

more effective.  Also, we usually incubate all reagents for at least 5 h or
overnight, and wash several times over the course of two hours.  Also, all
antibodies do not penetrate equally well.  Some antibodies that work well on
sections, simply will not stain in whole mounts.  Probably it has to do with
the charge of the antibody or the location of the antigen.  Also, I find that
the fluorescent tyramide HRP substrates provide a nice consistent signal
throughout the tissue, whereas fluorescent secondaries or HRP/DAB staining
sometimes results in inconsistent or spotty staining.

Good luck.

Karen Larison - University of Oregon

----------------------------
Date: 15 Feb 1999 19:00:14 -0600
From: klosen <@t> neurochem.u-strasbg.fr (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.