RE: [Histonet] Question about De-calcifying mouse paws (long)

From:"Elizabeth Chlipala"


Gayle is correct especially about fixation.  We like to fix for a
minimum of 48 hours, placement of specimen in decal prior to adequate
fixation can cause extremely poor results.  Depending upon how you
section your mouse joints (we normally have 8 joints per animal, 2
knees, 2 ankles, 2 front paws and 2 rear paws) once we receive the
joints, prior to decalcification we trim the knees to remove most of the
muscle and trim the ankle from the rear paw and trim the front paws to
the size we need.  If this can be done by the individuals at necropsy it
will aid in fixation, if not then we do as soon as we get the specimens,
if we feel that they have been adequately fixed then we place the
tissues in decal.  We process two joints, sometimes four depending upon
the client per block.  If I had my way it would be no more than two like
tissues (ie two ankles, etc) but even with the mouse model since the
disease is not symmetrical you might have one ankle that's swollen and
the other normal, ultimately on those blocks you are cutting two levels
so you can demonstrate what needs to be demonstrated.  We decal in the
cassette with 5 to 10% formic acid depending upon the time frame.  I
would use at least 10% formic acid, change frequently if you want to
decrease the time in decal.  Knees will take 48 hours but ankles and
paws take longer.  We never cut any of the tissue or skin.  Use of a
shaker is going to help decrease the decalcification time.  If you are
getting white spots in your samples then your decal solution is
saturated with calcium and is depositing back into the tissue, when you
stain them do those white spots appear purple?  Regardless of the time
in decal the day before we are going to process these specimens we
always change the decal one more time, rinse well before processing and
process on a cycle that is about 16 hours long, three changes in
absolute, and xylene and 4 paraffin baths.  We like to decal for about a
week ideally, but you can speed that up if necessary.  For the rat
samples the same applies, if you can get the necropsy techs to cut the
toes off at necropsy you will get better fixation.  Rat joints take a
while to decal, knees in 48 to 72 hours and ankles 5 to 7 days, but you
can speed it up with more frequent changes of decal.  


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
P.O. Box 18592
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
Ship to Address:
Premier Laboratory
University of Colorado
MCDB, Room A3B40
Boulder, Colorado 80309

-----Original Message-----
[] On Behalf Of Jamie E
Sent: Thursday, November 17, 2005 12:46 PM
Subject: [Histonet] Question about De-calcifying mouse paws

HI All,
           Here is my problem, We are a research  histology lab which 
supports groups doing Mouse/ Rat Collagen Induced Arthritis (CIA). The 
researchers collected the paws and knees for routine processing
and we take it from there. We are trying to give a quick turn around
(2 weeks) on these studies so we are fixing the paws for 24 hours in 10%

neutral buffered formalin then switching it to CAL RITE (a 
formaldehyde/Methanol/ Formic acid mixture from Richard Allan) for 
decalcification. The paws sit in Cal-Rite for 2 days on a shaker and are

then trimmed by cutting the skin and one toe on each side off the paws
trimming 1/3 off the knee Sagittal section (knee and some of the long 
bones), this helps in decaling the paws and knee quicker. Then the
are put into fresh De-cal again for 2 more days before  washing for 1
in tap water and processing. 
Problem: Knees are great , section great look great but some but not all

of the paws are chalky, white deposits in toes and ankles. This only
happens to about 1/3 of the 
samples. I guess they are not de-calcified long enough? 
Is there another way people are De-caling quickly with better results?
Pathologist is happy with the slides for the most part but the bad ones 
are more difficult to section as you can imagine and sometimes the ankle

joints don't section well at all. Any Ideas would be greatly


Jamie Erickson
Sr. Research Associate 
Department: DSMP
Abbott Bioresearch Center
100 Research Drive
Worcester, MA 01605-4341
FAX: 508-793-4895
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