[Histonet] cryosectioning adult fish teeth?

[Josh Trapani]

Dear All,

I have been attempting to cryosection pharyngeal teeth (and associated
structures) of adult fish for immunohistochemistry, with very limited
success. I have been working mostly with zebrafish but also with some
cichlids; adult body lengths are < 5 cm. The problem is that even when the
section as a whole is good, the tissue within it contracts, folds, and
just doesn't hold together. The protocol I am currently using is as
follows:

-Immediately after death, branchial arches are dissected out and fixed
overnight in 4% paraformaldehyde in PBS at 4 C.

-Fixed material is then transferred to PolyNoCal (Polysciences);
decalcification takes < 24 h at 4 C.

-Material is then transferred to 30% sucrose solution and left overnight
or until the specimen sinks...this also at 4 C.

-Finally, material is embedded in Tissue-Tek OCT, frozen carefully in
liquid nitrogen, mounted and cut at -20 C. Optimally, I would like
sections of 15 microns thickness.

Some variations I have tried that don't improve things much include:
sectioning whole heads instead of dissecting out branchial arches, longer
fixation times, decalcification in 4% EDTA (takes a lot longer), freezing
of specimens in the cryostat just prior to sectioning rather than with
liquid nitrogen beforehand, and cutting sections up to 30 microns thick
(thicker sections do generally hold together better, but it's still not
too good).

Searches of the literature have not helped me come up with a better
protocol. Something tells me if I had more histological experience, the
solution would be obvious, but I may be wrong. My best guess right now
is some incompatibility between the tissue and the freezing medium, so I'm
looking into alternative freezing media.

If anyone has any suggestions or knows of a protocol that works (for
cryosectioning mineralized tissues in adult fish, or any lower
vertebrates really), I would be very appreciative.

My apologies if this question has come up before (though somehow I doubt
it!).

Josh Trapani
Postdoctoral Research Associate
Department of Ecology and Evolutionary Biology
University of Colorado
Boulder, CO 80309-0334