[Histonet] cryo preserving and sectioning of bone

[Gayle Callis]

One young fellow has fixed murine nasal turbinates in PLP overnight at 4C,
changed fixative and fixed overnight again.  It is advisable to perfuse the
animal with PLP at euthanasia via heart before immersion into larger vol of
PLP. 

After PLP fixation, he decalcifies in tetrasodium EDTA (1.25%) dissolved in
20% sucrose in Dulbecco's PBS.  He adjusts the pH to 7 or so.
Decalcification is done at 4C after drawing a vacuum to exclude air bubbles
in nasal passages and allow EDTA better, close contact with turbinates.  It
can take up to 1 week or longer to decalcify.  Snap freeze using a petri
dish floating in layer of liquid nitrogen.  Embed bone in OCT in a cryomold
(Peel a way, plastic cryomold) and sit this inside petri dish until block
is frozen.  DO NOT LET LIQUID nitrogen get into petri dish, just let dish
canoe in this with platform support, don't want mold to tip over into Liq
N2. which can crack block from extreme cold.  The EDTA decalcified bone
cryosections without problems with excellent immunostaining. 

Advantage, decalcification while cryoprotecting with sucrose solution.  We
have one project (hamster turbinates, PLP fixed overnight, with EDTA upped
to 5% concentration in 20% sucrose in DPBS, adjusted to pH 7.6 with glacial
acetic acid. We chose to raise the pH a bit to speed up decalcification
since EDTA decalcifies as a function of pH, with lower pH giving slower
decalcification rate but staying within a working pH similar to buffers
used during immunostaining - pH 7 to 7.6.  

Acid decalcification is avoided although should work IF one rinses acid out
thoroughly and your antigens survive acid exposure. 20% to 30% sucrose
cryoprotection will help rinse out any residual acid, 2 changes might even
be better. One could also do tedious acid neutralization.  We don't like
the idea of residual acid in section trimmings sitting around inside the
cryostat for potential corrosion of metal parts. 

We do a simple decalcification endpoint check, weight loss, weight gain
method that puts us in the ball park for total calcium removal, it works
with EDTA quite nicely and acid decalcification methods.  Hopefully you
have a FAXITRON for sensitive endpoint determinations.  EDTA chemical
determination is a pain to do, although a good idea with acid methods. 

 
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)