We always do our PFA fixation in refrigerator, but is 48 hours enough for
even the dense bone of tiny mouse tibia, hopefully in large volume.  The
variables in this system are cold temp fixation with PFA fixation before
EDTA.  I would bet your fixation is being completed in alcohols of
processing - a hardening experience for bone!  EDTA has been used to
decalcify UNFIXED mouse bones for cryomicrotomy, with frozen sections cut
after this is done, then acetone fixed -  SO if your bones are not well
fixed in cold PFA, they will still be decalcified effectively by EDTA, then
you go to alcohols. 

You could try longer cold fixation to see if this counteracts your problems.  

Once it is TOTALLY fixed, I don't see why EDTA couldn't be done at room
temperature, although it may not be affected much by temperature since it
is a chelating agent.   

Next question, do you do ISH on your normal fix/decalcification with
excellent results? If so, I would point out your way works better with
identical results, give this person some side by side comparisons. 

Good luck

 

At 09:40 AM 10/30/01 -0500, you wrote:
>	I need a pressing question answered. I am working with someone that
>insists that fixation and decalcification should be done at degrees for ISH.
>
>
>We are fixing mouse tibia in 4% PFA for 48 hours at 4 degrees and decaling
>in 10% EDTA for 14 days at 4 degrees both on a rocker with several changes.
>
>I use formalin for 48 hours at RT and 10% EDTA for 14 days at RT for similar
>projects and have no problems. The stuff for ISH is dry, brittle and hard to
>cut. 
>
>Is it really necessary to do fixation and decalcification in the refrig? I
>am at my wits end!!
>
>Thanks
>
>Jennifer Harvey
>University of Rochester
>
>
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)