[Histonet] Re: OCT embedding problems

gayle callis gayle.callis <@t> bresnan.net
Tue May 13 11:40:18 CDT 2014

You Wrote: 


Hi everyone.


With Snap frozen tissue in OCT.  If I cut sections and fix them in cold
acetone for 10 minutes, (Is 2 minutes vs 10 minutes going to cause any


Is it better to dry them and then put them in DPBS or just put them straight
into DPBS?


If I changed the fixative to 10% NBF would that be better for the tissues
than Cold Acetone?


I am still having problems with my Frozen sections looking digested, and I
am not really sure what is causing it.





You never said how you store your frozen section immediately after
sectioning  and before acetone fixation?   You should NOT store frozen
sections in a cryostat, a thaw/refreeze issue.  Also, when you take the FS
out of the cryostat, water condensation forms, and this alone can damage the
morphology along with antigens.  It is better to section and dry at RT.
Seattle can have a very humid air, and that means more water condensation
formation if you store your sections in the cryostat before acetone
fixation.   Drying in front of a fan is ideal.  


We had much better success with air drying frozen sections at RT BEFORE a
minimum of 30 minutes going into 4C acetone for 10 minutes.   Fixation times
and temperature of acetone varies from lab to lab.   Fixation time in cold
acetone also depends on the antigen, some antigens do better with longer
fixation at 10 min and/or longer drying at RT.   We  fixed human renal
biopsy sections for 10 dips (approximately 10 to 15 seconds)in 4C acetone
when doing  immunuglobulin immunofluorescence staining.   The sections were
always air dried before fixation a minimum of 30 minutes or  longer.  After
acetone fixation, the sections sit for 15  or so to allow  acetone to
evaporate before staining or storage at -80C.  We found water was the enemy
with frozen sections destined for solvent fixation.  


However, if you are going to stain with an H&E or for antigens that
withstand NBF fixation, then you can put the section immediately into RT NBF
for an hour to overnight, or some set optimized time.   Remember NBF needs
time to the crosslinking of proteins, and 1 minute may not be ideal .
Since it takes a bit of time to do the actual sectioning, by the time you
finish with the last section, the first one immersed in NBF may be
adequately fixed but keep track of approximate time.   It is a good idea to
optimize NBF fixation time.   With longer NBF fixation and doing an H&E
stain, the longer it fixed the better the section looked, plus was stable on
the slide (no lift off).   Overnight NBF fixation was popular for H&E if it
wasn't for rapid diagnosis.   Our frozens looked very much like FFPE unless
there was incorrect, slow snap freeze causing freezing artifact.     


Gayle Callis


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