RE: Inadequately fixed bone

From:"Elizabeth Chlipala"


When you received the guinea pig joints were the knee caps removed and
the majority of the muscle removed from the femur and tibia.  We
normally allow guinea pig joints to fix for 48 hours and then decal in
5-10% formic acid, when they are received like I described.  They might
be over decaled.  It shouId not take longer than a week to decal guinea
pig joints in 5% formic acid, usually the joints are decaled well enough
to gross in (cut along the frontal plane) in 72 hours, if you change the
decal solution daily.  If these joints are from an OA study and you are
looking for cartilage degeneration, you might be o.k.  From my
experience if you are running a toluidine blue stain, slight over
decalcification does not effect the toluidine blue staining of the
articular cartilage.  Extensive over decalcification will cause loss of
toluidine blue staining.  What I mean by extensive is when you would
stain a section with H&E, the nuclei would stain red.

I have seen what you have described also and normally the articular
cartilage is still fine.  If you feel that the sections are decaled
enough I would process, cut and stain a few just to see what they look

Good Luck


Elizabeth A. Chlipala, BS, HTL(ASCP)
Premier Histology Laboratory, LLC
P.O. Box 18592 
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
Ship to Address:
Premier Histology Laboratory
University of Colorado
MCBD, Room A3B40
Boulder, Colorado 80309
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-----Original Message-----
From: Chan Wai Kam [] 
Sent: Friday, August 15, 2003 2:28 AM
To: HistoNet Server
Subject: Inadequately fixed bone

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.

Julee C
Orthopaedic Surgery
National University of Singapore
E-mail :  

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