Z-fish and TEM questions

kingtc at uwm.edu kingtc at uwm.edu
Mon Mar 5 11:02:17 EST 2001


I am a gradutate student at the University of Wisconsin, Milwaukee 
and am taking a course in TEM.  For my project I plan to look at 24 
hr zebrafish embyros that have been exposed to ethanol, and look 
for any morphological anomalies of the brain.  I looked through the 
methods posted on the internet, but did not find any pertaining 
directly to TEM.  I was hoping you could give me some advice 
(unfortunately, my advisor has not done TEM with zebrafish and my 
instructor works with plants).  

Can you suggest a good fixation protocol?  I have found several 
fixation "protocols" from papers, many of which are mixtures of 
gluteraldehyde and paraformaldehyde in either phosphate or 
cacodylate buffer.  On the web, for general fixation,  glut and 
paraform is suggested, but is this for EM or LM? Can you give me 
an estimate to as how long I should fix the embryos and whether it 
would be best to embed the embyros in agar for easy 
manipulation?  

I plan to infiltrate in Epon.  Do you have any suggestions as to the 
timing for infiltration, or if there is a better plastic I should use?  I 
plan to stain sections with uranyl acetate and lead citrate.  

Thank you for your time.  I greatly appreciate any advice you can 
give me.  

Sincerely, Tisha King Heiden








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