Why are the cryosections fragmented and how can this be solved?

Klenow bakedbeans at spam.not
Wed Mar 3 00:50:59 EST 2004


Un-cryoprotected tissue fixed with 4% paraformaldehyde shatters easily
making it difficult to get good sections.
As one of the other posters said, cryoprotection with sucrose will help
reduce ice crystal formation as will very rapid freezing.  You might also
try dipping the tissue in chilled (-30 to -40 deg. C) isopentane on dry ice
for rapid freezing.  Rapid thawing of the sections is also preferable...e.g.
thaw-mounting directly onto coated slides or thawing in room temperature
saline and then slide mounting.

"horseboy" <qc00 at mails.tsinghua.edu.cn> wrote in message
news:b208bea0.0403020505.7c1e381 at posting.google.com...
> I was doing a cryosection and X-gal staining of Drosophila brain
> section. At first I fixed the whole head in 4% paraformaldehyde for
> about 4h,make the freezing sections and stain in X-gal overnight at
> 37oC, but the staining result is not good; this time I tried to fix
> the head for 0.5h,1.5h and 3h respectively and the staining result of
> 0.5h was good but most sections are fragmented.
>
> It seems that too long fixation time will destroy the enzyme activity,
> but short time may results in fragile tissues. How can sections be
> kept intact under the condition of short fixation time? Can we achieve
> this by prolonging the freezing time or lowering the freezing temp.?
>
> Looking forward to your answers. Thank you.






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