[Histonet] thick sections in zebrafish

From:"Eric Tytell"

Hi all --
  I'm trying to get thick (between 50 and 100 micron) transverse sections of
adult zebrafish (15 to 20mm long).  I'm concerned about the 3D structure of
the muscle tissue, so I'd like to have the specimens embedded in some
supporting medium.  I've had good luck using plastic (JB-4) sectioned with a
saw, but the trouble with that method is that I lose 300microns to the saw
each time I cut.  I'd like to try embedding in a different medium, but I
haven't been able to find a protocol.  Has anyone tried to do something like

I'm currently experimenting with paraffin embedding.  Specifically, does
anyone have suggestions for how long and in what to decalcify?  I currently
have both EDTA and Poly-NoCal available.  Also, how long should I infiltrate
in paraffin?

Alternatively, I've tried using a cryostat microtome, but the muscle tissue
seems to be damaged by the freezing or possibly by desiccation.  Is it
necessary/possible to decalcify for frozen sections?  Any suggestions for
avoiding freezing damage?

Also, does anyone know of a good polyclonal antibody for zebrafish skeletal
muscle?  I'm not looking for anything too specific -- just enough to
distinguish skeletal muscle from bone and collagen under low magnification
confocal imaging.

I'd appreciate any suggestions.  Thanks in advance,
Eric Tytell

Eric Tytell
Museum of Comparative Zoology Laboratories
Harvard University

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