Terry,

We do a lot of work with zebrafish embryos here.  Almost all the ICC we do is
on frozen sections or whole mounts.  We avoid paraffin work for a couple of
reasons: (1)  Because we're working in fish, a limited number of antibodies
are
available, and most of these are morely likely to work on frozen sections, and
(2) it's difficult to align the very small embryo in hot paraffin.  Instead we
align our fish in a hot agar/sucrose solution, and this enables us to create a
block with the embryo aligned for the correct plane of cryosectioning.

Most of the postdocs here, however, prefer working with whole mounts for both
ICC and in situs.  So we have procedures for permeabilization, etc.  For ICC,
we commonly use either fluorescent secondaries, or PAP
(peroxidase-antiperoxidase) methods; Vector's ABC complexes are simply too
large to penetrate the embryo well.  HRP/DAB methods are commonly used for
ICC.
I've also found that fluorescent tyramide HRP substrates from Dupont NEN work
extremely well with whole mounts, providing a much more sensitive and
consistent signal than DAB.  For in situs, we use alk phos-mediated methods;
the best substrate is NBT/BCIP.  People sometimes double-label using
fluorescein- and digoxigenin-labeled probes.  The second alk phos substrate
used in this application is Fast Red, and stripping methods must be used
before
the second Fast Red signal is developed.  However, the Fast Red signal has
much
poorer resolution and is soluble in organics.  Although I've tried other
substrates such as New Fuchsin, these tend to be absorbed by the yolk.
Although the yolk can be removed, it's best to avoid tedious microsurgery if
one can avoid it.  Some people also use the HRP/DAB methods to develop the
second signal, thereby avoiding the stripping step.  However, HRP/DAB is far
less sensitive than alk phos-mediated methods, and can be used only with very
robust probes.  Commonly, the stained embryo is then sectioned to clearly
elucidate cellular details.

If the researcher requires good detailed cellular morphology, we embed the
embryo in epon and cut 5 um sections on a glass knife.  The DAB signal
survives
the epon processing well.  However, the NBT/BCIP signal is slightly soluble in
organics; the less hydrophobic methanol is used for dehydration and the
dehydration/infiltration schedule is shortened considerably.

I hope this helps.  Please don't hesitate to contact me if you have questions.

Karen D. Larison

Date:          Wed, 06 Jan 1999 15:50:14 +0000
From:          Terry Hacker <T.Hacker@har.mrc.ac.uk>
To:            'Histonet' <Histonet@pathology.swmed.edu>

Does anyone know of any seminars/workshops coming up on
the subject of embryology, including the development of the embryo
( particularly gonads ) and also Histological procedures ( paraffin,
frozens for ICC , ISH etc. ).
I have worked mostly in the area of oncology but now the work is
moving towards Genetics and Embryology, and dealing with tiny 7-10
day embryos is quite different from adult tissues.
Alternatively, are there any experts out there willing to give some
advice?
Many thanks, Terry.

Terry Hacker,
Medical Research Council,
Harwell,
Oxfordshire,
U.K.