Hi everyone... been having some trouble lately with my
cryopreservation. Tissue looks grossly fine before embedding in OCT but
then when I cut sections on the crystat the architecture is totally
disrupted with a lot of space in between cells. It almost looks like
the cells have become so shrunken and dehydrated that they actually
pull apart the interstitium.
Has anyone ever had this problem? Can I skip the sucrose and embed after PFA
(paraformaldehyde) fixation...will that still preserve my signal?
Here are some additional details about my protocol, which has always worked in
the past so I'm not sure why its a problem now:
-this is adult tissue (kidney, liver, tumor, etc) containing an endogenous YFP
reporter that needs to be preserved
-tissue is fixed in 4% PFA overnight at 4C, then in 30% sucrose/PBS overnight
at 4C
-once
tissue has sunk in sucrose soln (typically next day), I let tissue sit
on paper towel for a few sec to dry up sucrose soln then let sit in OCT
for a few min before placing into OCT boat and slowly freezing on dry
ice.
Any suggestions would be very much appreciated...thanks so much for your time!
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