Quick reply -

I frequently cool my knife with dry ice or with that freezing spray helps
sometimes.

Just need to keep trying sometimes.

c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317

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-----Original Message-----
From: Joanne Whitehead [mailto:joannew@bluebottle.com] 
Sent: Monday, October 03, 2005 2:04 AM
To: histonet@lists.utsouthwestern.edu
Subject: RE: [Histonet] "empty" cryosections!

First of all, many thanks to Gayle and everyone else who has offered
suggestions on freezing and sectioning.

I am using a Jung CM3000 cryostat with disposable blades. The machine
is showing it's age - it has needed several visits from the Leica
technicians lately. But I have just installed a new antiroll plate,
which is a bit of help. I have tried the brush technique also, but I
can see as the tissue comes across the knife it is already pulling
away from the OTC, so this is not an issue of the antiroll plate. I
hadn't considered variations in temperature within the machine - I
will check for that next time. I wondered if it would help if the
knife was colder than the tissue - anyone tried this? A couple of
pellets of dry ice on the knife holder perhaps? 

I will definitely try embedding the tissue in OTC before freezing next
time, as well as infusing with OTC before freezing. But I wonder how
to make a suitable mold? Someone has suggested aluminum foil. I
ususally use re-useable plastic rings if the tissue is small enough,
or else cut a piece of the bulb from a disposable plastic pasteur
pipette for larger pieces, which I can just cut away after freezing.
Also I understand from the comments here that mixing dry ice directly
into the isopentane, rather than having the isopentance suspended
above liquid nitrogen will give a more consistent temperature for
snap freezing - another modification I will try next time.

I had already settled on -26C for sectioning, as suggested by Gayle. I
need to open up the intestine for my manipulations, so infusing the
tube directly with OTC is not an option for me. But I need sections
only 1 cm long, so it rolls up into a reasonably compact form. I have
been rolling with villi out so they will be spread apart better, but I
have also seen images rolled villi in - any preferences out there?

So thanks everyone, hopefully my next attempts at freezing &
sectioning will go much smoother. That brings me to my next problem,
which I will describe in a separate post :)


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