[Histonet] Re: Cutting frozen fat

From:Linda Jenkins

         Your message read:

"Hi all:    I have a project that the researcher wants
frozen sections on mouse fat for immunofluorescence.
I froze mesenteric and epididymal fat at necropsy in
isopentane.  I am having a hard time getting the
tissue to cut-I'm getting the hole in the middle of
the OCT block.  I have changed the temp in the
cryostat to range from -16 to -35 and inbetween to see
if colder would help.  Not much luck. Does anyone know
if I can defrost and fix the tissue and will it
improve the cutting or will that cause it to loose any
FITC. Thanks for any suggestions.  Pam"

First of all...How did you store the specimens until cryosectioning?  If 
you used a -80C freezer, I have found that it is useful to allow 1/2 day 
for acclimating to the -30C cryostat temp.  Also, for holes in the sample, 
I use a wooden tongue depressor which has a drop of fresh freezing compound 
(OCT, etc) placed on one end and, using a "spackling" technique, I quickly 
fill the hole with fresh freezing compound and allow it to 
refreeze.  Remember to back off on your knife setting as this will increase 
the height of the sample.
         Happy cutting!

Linda Jenkins, HT
Clemson University
Dept. of Bioengineering
Clemson, SC 29634-0905

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