If your bones are not totally fixed and exposed to that high concentration
of acid, your bone and soft tissue/cells undergo protein hydrolysis, very
damaging to your tissues and they will look horrible, probably section
badly too. You could reverse it, but the damage is already done by the acid
- that is why they are soft (calcium gone) but still unfixed - not a good
thing to have happen. Even if you did salvage the samples, the tissue and
subsequent staining will be poor.  

Things that help bone fixation:  

If you can, bisect the bones or drill holes away from the area you want to
examine to allow fixative penetration into bone which fixes very slowly due
to calcified bone matrix. If you are not going to look at the whole bone
i.e. femur, tibia, head - cut away any bone you don't need or want to
examine. The smaller and more open a larger bone sample, the faster it
fixes.  This rule also applies to decalcification. 30% formic acid is very
strong, and hopefully you control your decalcification with endpoint checks? 

Suspend bones in NBF and use a HUGE volume, at least 20 times fixative to
volume of each bone.  Change the fixative after a day or so to replenish
active fixing agent (formaldehyde) and let these larger bones fix longer.
If is always good practice to change the fixative again about halfway
through, say 5 days and let them fix another 5 days. 

When working with a new species, we take one animal as a pilot study and
bisect bone of choice i.e. femur, tibia or whatever at 10 days, if it still
pink inside, you KNOW you must fix longer.  That way we don't mess up the
experimental group and eliminates guessing fixation time needed.   Once we
determine how long it takes to fix, we suspend experimental group bones in
bags into a couple of liters (if not more) of NBF. 
Fixative is cheap so we use a huge amount for bone work. 

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

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

email: gcallis@montana.edu


   At 05:28 PM 8/15/2003 +0800, you wrote:
>Hi Histonetters,
>
>I've not encountered this problem before so need some advice.  I am
>processing some guinea pig knee joints.  I placed them in NBF for a week
>and they are now decalcifying in 30% formic acid.  From their appearance
>(still slightly reddish and soft), I suspect they are not properly
>fixed. Fixed tissues usually appear compact and yellowish. Is it
>possible for me to reverse the procedure back to fixation again or are
>my specimens beyond salvaging?  Would appreciate your advice.
>
>Thanks
>Julee C
>Orthopaedic Surgery
>National University of Singapore
>E-mail :  doscwk@nus.edu.sg  
>
>
>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

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

email: gcallis@montana.edu