[Histonet] formalin fixed tonsil frozen sections

[Gayle Callis]

Dear Erin 

 

You wrote: 

 

Good morning!  My pathologists would like us to cut formalin fixed (not yet
processed) tonsil tissue on a cryostat for DIF staining.  Has anyone done
this?  I did a quick search that seemed to indicate that it was possible but
that the architecture of the cells would be altered because of ice crystals
and that it would be difficult to get the sections to stick.  If you have
any advice I would greatly appreciate it!

 

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Several things to think about: 

 

FF tonsil should be cryoprotected with 20 -30% sucrose before snap freezing
to prevent ice crystal formation.  Simply take the FF tonsil, cut into
smaller pieces and immerse into 30% sucrose over night at 4C.  When the
tissue sinks to the bottom of this somewhat thick syrupy solution as which
indicates the tissue is cryoprotected.  Blot off excess sucrose, embed in
OCT and snap freeze.  You can snap freeze many blocks to stockpile controls
and store these at -80C, shorter time at -27C.  

 

One thing you did not mention is:  are tissues you are trying to do DIF on
for diagnosis also formalin fixed?  I would think a tissue control should be
handled the same way as the test tissue i.e. fresh tissue frozen sections
fixed with cold acetone compared to FF tissue.  If your tissues are not FF,
then why not collect fresh tonsil and snap freeze that as a control?  You
can make several blocks and store a -80C or even -27C (for a short time).
The concentration of your antibodies may be different on a FF tonsil section
due to the cross linking as compared to the concentration on a fresh tissue
frozen section fixed with cold acetone.   Antibody dilution would have to be
tested.  Personally, I would want my antibody concentration for a control
and test tissue to be the same.   If you already run DIF on a FF tissue from
patient, then a FF control works fine.   

 

If you are using FF tonsil sections as controls, then FF tissue will have
aldehyde induced autofluorescence but this can be removed.  Autofluorescence
is annoying when trying to read the sections and could mask what you want to
see unless you use a contrasting color fluorophore.    

 

To get sections to stick, mount frozen section on reliable plus charge
slides, and air dry the frozen section a bit but be gentle when rinsing the
sections.  Formalin fixed frozen sections always have the possibility of
coming off, but many have success retaining the section and discussed many
times on Histonet.    There may even be newer plus charge slides touted for
FF fixed frozen sections these days.  

 

Good luck

 

Gayle M. Callis HTL/HT/MT(ASCP)