[Histonet] "empty" cryosections!
joannew <@t> bluebottle.com
Mon Oct 3 04:03:50 CDT 2005
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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