We always used NBF as fixative, and it must be noted that alcohol can slow
down decalcification, that is why it is added to special decalcifying
solutions (discussed in Lillies book). Your sample will go through
rehydration in the combination decalcifier, which takes time so maybe the
alcohol is not even needed.      

We then went into a decalcifier, your laboratory choice, formic acid is a
bit slower or rapid decalcifier, for 1 hour, rinsed and processed, but a
biopsy was TOTALLY fixed before decalcification step, as yours is - my
compliments!   

The important thing it to insure bone is totally fixed, so why not use just
the formalin/formic acid combination - it may do the job all in one step
with rate of decalcification never exceeding rate of fixation. OR just fix
in NBF and then go to your combination fixative/decalcifier. I would tend
to opt for fixation first, then decalcification.  We used to do one hour
NBF, preferrably longer, then decalcify for one hour.  

I also know one lab where a pathologist kindly dissected off the cortical
bone plug often found at the end of a trephine biopsy - it speeded up
decalcification, got rid of excess bone that created the residual calcium
problem. 

Just some thoughts 


Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu