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

kenneth p Collins kpaulc at earthlink.net
Tue Mar 2 16:56:15 EST 2004


"kenneth p Collins" <kpaulc at earthlink.net> wrote in message
news:Bm71c.30123$hm4.20253 at newsread3.news.atl.earthlink.net...
> "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.
>
> I'm not 'familiar' with your problem, but suggest
> that you perfect your sectioning technique with
> untreated tissue - you know, separate the var-
> iables, establish your expertise with the simpler
> problem, then add variables, proceding only when
> you're satisfied with the results at each level of
> complexity.

What I wrote is inadequate.

=Freeze= and take sections without otherwise
treating the tissue.

Then add other variables, one at a time.

ken





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