[Histonet] First time with frozen sections, long reply

[Gayle Callis]

You wrote:
I am starting to stain frozen sections for the first time. Until now I
have only stained paraffin embedded sections and so was hoping for some
pointers.I contacted our histopathology lab who provided me with the following
information: They don't use any antigen retrieval method.
They pretreat the slides only with methanol for 10min followed by drying
before staining. Then they proceed with the normal steps of staining and 
dehydration.
I have however read that acetone can be used instead of the methanol.
What is the difference that this provides? I am trying to stain for Stat5.
Is there anything els I should keep in mind when handling frozen sections?

Questions:

human tissue, animal tissue? Is the tissue fresh, unfixed or prefixed prior 
to snap freezing?

Have you snap frozen (needed to minimize freezing artifact from water ice 
crystal formation) the tissues rather than cryostat freezing (too warm)?

What antigens do you want to stain for?

Fixation is dependent on the antigen, some will be fine with acetone, some 
may work better with another fixative.

Antigen retrieval on frozen sections of fresh unfixed tissues is not needed 
as long a formalin or even paraformaldehyde is avoided.  In general, AR 
tends to damage FS.

If you plan to do CD marker staining, methanol can cause poor staining of 
these due to methanolic bridge formation.  Avoid methanol as a fixative or 
in peroxidase block, use DAKO S2002 peroxidase block designed for gentle 
endog perox blocking, it doesn't contain MEOH and wil not chew section off 
the slide.  Hydrogen peroxide can be put into PBS instead.

Antibodies also need to be optimized since FS permit lower conc of 
antibodies.   Dilution panels should be done.

What does the spec sheet or manufacturer recommend for fixation of FS for 
your STAT5 antibody?  You can always call their tech services and ask.

Be gentle, FS are more fragile, and air dry them AFTER sectioning, DO NOT 
STORE newly cut setions in a cryostat. Avoid water condensation and freeze 
thawing of sections - this can damage antigens.   Air drying overnight in 
front of a fan, or over dessicant is a good way to insure properly dried 
sections.

Check Histonet Archives www.histosearch.org, it has tons of info on HOW to 
do frozen sections, fixation, blocking, and staining. Chris van der Loos 
has provided many excellent replies on frozen section IHC. DakoCytomation 
website has Immunochemical Staining Methods Handbook, 3rd Edition with tons 
of hints on what you want to do.

Good luck





Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)