Dear Sharon,
   I've done a lot of free-floating sections in the last few years and I think dealing with free-floating sections is much more difficult and time consuming than dealing with sections on slides.   I agree with Rob that the sections are sticking together because they are being fixed together.  They would need to be fixed separately and then combined together if needed.  
  I would suggest that they mount their unfixed tissue to slides, fix, and proceed with the staining.  


Sarah Christo, HT (ASCP)
Texas A&M University
College of Veterinary Medicine
Dept. of Vet. Anatomy & Public Health
College Station, TX  77868-4458
schristo@cvm.tamu.edu

>>> Sharon E Horton <sharon.horton@sri.com> 07/13 2:19 PM >>>
Dear Histonetters,

    I recently had a colleague ask a question I could not regarding
fresh frozen floating sections.  I'm passing it on to you in hopes that
you have more experience with floating sections than I do.  Any
suggestions or insight would be greatly appreciated.

Thanks
SH

forwarded message**************************************

Dear Sharon
I would appreciate if you could give me some advice (since I have tried
it
once but... )
My problem is that I was not able to perfuse the experimental animals. I

therefore have to do it on fresh frozen tissue. For what I read, people
usually collect the sections on slides and process to the in situ. I am
currently working on finding a way of fixing the tissue to be able to
work
on free-floating sections. I believe the sensitivity would be greater
and
it would be easier!

I have tried different things. I (and a technician in my lab)
collected sections in :
- 0.5% paraformaldehyde or,
- PBS 1M or,
- CBS (glycerol-etc...)
The sections were rinced once (and still looked fine).
Then, we fixed the sections for 2hrs in 4% Paraformaldehyde.
That's when it went wrong!!!! The sections sticked to each other and it
was
impossible to work with them.
Do you have any idea of what went wrong? of what I should do to make it
work?

Have you tried to work on free-floating sections from fresh frozen
tissue?
Do you know if people have tried before? Do you think that it's even
possible or am I wasting my time?

 I would really appreciate your help.