Hi Eric,
      I have read your question with some curiosity. If the JB4 supports
your specimen without distortion, why not use a motorized  rotary microtome
to section? If you have any questions, please feel free to phone me.

Don Birgerson
Leica Microsystems
Technical Assistance Center
Don.Birgerson@Leica-Microsystems.Com
1-800-248-0123  ext 5918   7:00 - 4:00 CDT


                                                                                                                                                    
                      "Eric Tytell"                                                                                                                 
                                    To:                                                  
                      Sent by:                              cc:                                                                                     
                      histonet-bounces@lists.utsouth        Subject:  [Histonet] thick sections in zebrafish                                        
                      western.edu                                                                                                                   
                                                                                                                                                    
                                                                                                                                                    
                      04/28/2004 02:40 PM                                                                                                           
                                                                                                                                                    
                                                                                                                                                    




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
this?

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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