[Histonet] cutting frozen rat turbs,, a reply

[Gayle Callis]

We cut decalcified turbinates without using Cryojane.  Turbinates are
decalcified with (from 1.25% up to 14% tetrasodium EDTA in Dulbeccos PBS
containing 20% sucrose, pH is adjusted to 7.6  You could use formic acid IF
you rinse turbinates well and cryoprotect after acid solution. EDTA
decalcification is done at 4C, formic acid is done at RT. 

It takes a LONG time, and we do a weight loss,weight gain endpoint check
with mouse and hamster heads taking as long as 2 weeks, sometimes more.
Patience!   

If you decalcify with formic acid and after the water rinse,  you may need
to cryoprotect maybe a bit longer than overnight with 20 - 30% sucrose in
PBS at 4C to insure the cryoprotectant is infiltrated well into bone, etc.
I think you could even add OCT to this cryoprotectant mixture without
problems.  I helps to vacuum samples to pull out air bubbles in rat nose
before going to 4C.  Turbinates are snap frozen embedded in OCT by floating
a petri dish on a layer of liquid nitrogen, and using a Peel away mold, the
one that is 22 X 22, nose facing down to accomodate longer specimen.
Cryosectioning is done with a high profile AccuEdge blade - very sharp and
very sturdy. 

Sectioning is carried out at colder temps, -26C with sections picked up on
Plus charge, air dried at RT before IHC.  

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)