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Does anyone know how to avoid clumps formation during cryosection??

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Offline Rams

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cryosection urgent help
« on: September 09, 2011, 09:20:30 AM »
Hi all,

I've been trying to cut some cryosections of mouse spleen but I face with a recurring troubleshooting.
Whatever the protocol used to freeze the spleen in O.C.T (slow freezing using dry ice, snap freezing with liquid nitrogen in the presence or absence of isopentane etc..) I always see white clumps forming into the organ once cut with the cryostat. I tried to play with the temperature of the cryostat chamber  as well as the temperature of the knife but no way to avoid these strange clumps appearing.
I've never seen that in the past while I've cut many times this organ and I don't know whether these "holes" are due to the formation of ice crystals, but in the presence of isopentane I should not see them anymore?
I joined below a photo of the organ to illustrate the problem.


If anyone had any suggestion, protocol or idea to help me solving the problem, I would appreciate a lot!!

Thanky ou very much

Rams

cryosection urgent help
« on: September 09, 2011, 09:20:30 AM »

Offline GWinn

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Re: cryosection urgent help
« Reply #1 on: November 16, 2011, 07:00:22 AM »
Hi Rams,
Im not sure if Im on the right track here but.... you dont have an HE of the spleen to look at.
For a starter - Id freeze in Isopentane if you're interested in morphology, forget slow freezing and use LqNit as a second resort.  There are heaps of protocols but basically, use a disposable histo mold, fill with OCT, place in the tissue and freeze in your chilled isopentane, then store or cut sections.
Im not sure, but I suspect your white spots are lymphoid nodules in the spleen and these will appear paler than the surrounding splenic tissue. It would really help to see an HE.
Regards




Re: cryosection urgent help
« Reply #1 on: November 16, 2011, 07:00:22 AM »