[Histonet] Re: Mouse Paws

[Gayle Callis]

You processing schedule is short.  We process mouse paws NBF fixed for a 
week, decalcified with 10% formic acid using endpoint checks until paws are 
done - this can take up to 3 days in stronger formic acid.  Your buffered 
formic acid is approx 4.5% formic acid (did a calculation).  Our 
decalcifier is made from 88% stock formic acid.   Decalcification, 
surprisingly, can take longer than you think due to the tiny bones tightly 
packed inside skin with very thick pads,tendons, etc. There are some hints 
on how to do determinations to not have to test every paw you work with in 
a big study.  Of course, xray is the best method for endpoint 
determination, but not everyone has a FAXITRON.  However if you have this 
unit, use it for endpoint as it is the most sensitive endpoint 
determination and you can do ALL the paws with one shot of xray on an 8 X 
10 film.   If you can fix the paws so they stay FLAT with toes spread out 
-  this helps also.  When the toes curl up after dissection from 
radius/ulna or tibias, we have more problems.

We do 70%, 95% X 2, 100% x 2, xylene 1 change, Clearite 3 X 1 change, and 4 
changes of paraffin ALL reagents including paraffin are done with vacuum 
and pressure and only ambient temperatures.  ALL stations are 2 hours per 
station, very extended processing.   We found the density of these tiny 
paws required longer processing.   You could start with 1 1/2 hours per 
station, but if the problem still exists, up the times.  Our paraffin temp 
NEVER exceeds 60C.

Opaque, mushy tissue can mean several things. 1) Incomplete 
decalcification, 2) incomplete dehydration (mushy syndrome!) and paraffin 
infiltration i.e. processing or 3) both.

If animal has inflammed paws and you still have problems, then you can 
always add time to processing schedule.   We often cut open skin on the 
back of paws to allow better reagent penetration/echange.  Take care to not 
damage an area you want to see; this might work on pad side also.  Do this 
at collection, it is easier to slide fine point of scissors under unfixed 
skin and helps with fixation too. Use ultra fine point stainless steel 
cuticle scissors found in nail care aka glamour section of Walmart, Target, 
grocery/drugstores - made by LaCrosse and cost $7 to $9 (frequently on 
sale) - far cheaper than most vendors.

If you use Tissue Prep 2, a harder paraffin, this is a good selection for 
bone work for both infiltration and embedding.  We use it and find it works 
for soft tissues without sectioning problems.

Good luck


At 07:57 AM 12/3/2004, you wrote:
>Hello Histonetters,
>
>I'm looking for some help or guidance in obtaining good sections from
>mouse front paws, to include the bones of the wrist and digits, as well as
>maintaining the surrounding matrix.  This is an ongoing problem in our
>laboratory and we have a difficult time obtaining good sections at this
>level, the problem appearing to be inadequate infiltration of paraffin
>into the interior of the paws, leaving the center somewhat mushy & white.
>
>The feet are fixed in 10% NBF for up to a week, decalcified in formic
>acid/sodium citrate for 3 to 5 days, processed on a Shandon Pathcentre for
>45 minutes/reagent, 4 changes of paraffin, 45 minutes each, vacum &
>pressure on the waxes only.
>
>Any suggestions regarding fixation, decalcification, or processing
>enhancements to improve the overall picture would be extremely  helpful.
>
>Thanks!
>
>Tom Crowell
>BiogenIdec
>Cambridge, MA
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)