[Histonet] "empty" cryosections!

[Joanne Whitehead]

Hi everyone,
I'm relatively new at cryosectioning, and so far have found it to be a
very frustrating experience! Some days it goes very smoothly and I get
plenty of nice sections, but most often I feel like it's a battle of
wills with the cryostat, which ususally wins.

My major problem is getting "empty" sections - the media cuts smoothly
but the tissue itself curls or crumples up, leaving just an open
circle of media. The other difficulty is with sections wrinkling as
they come off the knife, instead of lying flat. Does anyone know why
this happens, or how I can prevent it? 

I'm sectioning mouse intestinal tissue, which has been rolled into a
"Swiss roll", fixed in paraformaldehyde, infused with sucrose, snap
frozen in isopentane over liquid nitrogen and embedded in OTC. I have
tried cutting at different temperatures, from -18C to -30C, with my
samples equilibrating in the machine at least an hour before I start.


I have seen protocols in which the tissue is embedded in OTC before
freezing, and also infusing the tissue with a mixture of sucrose and
OTC before embedding. Does anyone have any preference for these
methods?

I would really appreciate any advice!
Thank you!
 
Joanne
Curie Institute, Paris