Re: 100 micron floating sections

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From:"Karen D. Larison" <> (by way of histonet)
To:histonet <>
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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
(7 minutes) in ice-cold (-20C) acetone before the blocking step also helps.
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:          Mon, 15 Feb 1999 17:20:15 -0500 (EST)
Subject:       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

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