[Histonet] Cutting frozen fat

[Margaret Blount]

One thought on this subject: If you are setting up a lab with the knowledge
that you will be cryosectioning fat, consider buying a cryostat with twin
compressors, these will maintain much lower temperatures than single
compressor versions and will attain low temperature quicker, with the added
advantage that if one compressor fails, your cryostat will still work
normally. Just a thought for anyone considering a purchase. The twin
compressor cryostat I had came from Bright Instruments.

Regards

Margaret

Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 2QR  

-----Original Message-----
From: Due, Brice [mailto:BDUE <@t> PARTNERS.ORG]
Sent: Tuesday, March 15, 2005 4:07 PM
To: pam plumlee; HistoNet Server
Subject: RE: [Histonet] Cutting frozen fat


Hello Pam, I have successfully cut 10um sections of human muscle bxs with
end-stage myopathies where the muscle had been 99% replaced with fat. The
trick
as Alan Bright pointed out is to make sure nothing that touches the sections
is
warmer than the block.

Dry ice is my trick. I use pieces of dry ice to chill the block, stage,
blade,
and roll plates as I'm cutting. I use pelletized dry ice. If I am careful I
can
place several pieces on the stage/blade holder without them getting in the
way
of the roll plate. I leave one piece hanging over the blade edge. Between
sections I rub the roll plate with a piece of dry ice. My chuck holder
allows me
to place a piece of dry ice on top of the chuck without getting in the way
of
the blade. So basically I put dry ice everywhere and crank the chamber down
as
far as it will go. 

I usually cut 10um sections with a roll plate. The end-stage myopathy I
mentioned above had to be cut at 14um even with dry ice. The OCT in thinner
sections may not hold together well at dry ice temps. So you may have to
experiment with timings. How long you chill the block face or how long you
wait
after you remove the dry ice from the specimen before you start sectioning.
Sometimes I find that I get best results by chilling the block face and then
pressing my thumb to the block between sections. Playing with dry ice gives
you
access to temps from -79C up to the chamber temp. 

One further dry-ice trick: At if you have a small cup of dry ice in the
cryostat
you can chill your foreceps. At such low temps, you can actually handle OCT
sections with forceps. The sections do not stick to the forceps until your
hand
warms them up. I have used chilled foreceps to pick and place sections on
slides, and I have used the forceps in place of a brush when cutting with
the
"brush" technique.

Good luck!
-brice
Brigham & Women's Neuropathology 
Boston, MA



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of pam
plumlee
Sent: Monday, March 14, 2005 10:13 PM
To: HistoNet Server
Subject: [Histonet] Cutting frozen fat


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


		
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