[Histonet] Processing tissue engineering sample

From:"Kobler, James"

I would like advice about processing some experimental gels containing
fibroblasts.  The goal is to stain immunohistochemically for extracellular
collagen in small gel samples (5mm diameter, 300 micron thick).  It is desirable
to cut the gels perpendicular to the surface in order to get multiple sections
that can be stained with different antibodies.  Cryosectioning would be
preferable since dehydration would probably cause shrinkage of the gels.

Questions:  (1) should the gels be treated with a cryoperservative such as 30%
sucrose before freeezing? (2) any tips for orienting the gel discs for nice
cross-sections? (3) Is freezing in isopentane cooled with liquid nitrogen the
best freezing method?

Thanks very much,

Jim Kobler, Ph.D.
Mass General Hospital

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