Good morning Andi,
Probably not any more difficult than doing a whole mouse or rat skull other
than the time needed to complete decalcification.
We decalcify in 10% formic acid but do use a decalcification endpoint test
to make sure teeth and everything else are decalcified. They probably will
not take very long, so you may need to interupt the decalcification near
endpoint by placing in NBF overnight then resuming decalcification the next
day. You could use a mixture of 4% HCl/8% formic acid which is a bit
faster than the 10% formic acid, but don't let bones sit in this
overnight. If you want, I have a weight loss/weight gain method for
testing, which requires a balance the weighs in mg.
If you are worried about over-exposure to acid i.e. over decalcification,
and have the time on your side to have a good preparation, then 14%
tetrasodium EDTA with pH adjusted down to 7.4 will work also. That way you
don't need to worry so much about timing. We weigh out 14 g tetrasodium
EDTA, dissolve in 80 mls distilled water or Dulbeccos PBS, and adjust the
pH down with glacial acetic acid, then bring final volume to 100
mls. This is a method from Dr. Webb Jee, an orthopedic bone expert. We
have preference for the tetrasodium EDTA since it goes into solution so
easily and at that wonderfully high concentration versus EDTA or disodium
EDTA where these are only soluble to approx 10% with the help of heat
and/or addition of sodium hydroxide.
The problem with tetrasodium EDTA is the beginning pH is above 9, and
alkaline sensitive protein bonds will be affected by the high pH. We do
the pH adjustment with an electrode in the solution and titrate it to pH
7.4 as this will take a fairly large amount of acetic acid. I have
adjusted the pH to 7.6 which is also the pH of our TBS buffer, and achieved
excellent results. EDTA decalcifies as a function of pH and at pH 7 the
decalcification is slower since the molecule is not fully protonated until
pH 8. However pH 8 is pushing the limits for alkaline sensitive protein
bond related problems so we keep the pH around 7.4 - 7.6. I am going to
attach the weight gain/weight loss method to you privately - this works
with acids and EDTA methods, and was originally used for testing nitric
acid decalcification endpoint. If you have a FAXITRON, your endpoint
testing would be simple. EDTA is a pain to test chemically.
When processing, be sure to add some time onto paraffin infiltration with
these tiny dense bones plus teeth? As for sectioning, it will be easier for
bone if you use a harder paraffin i.e. Tissue Prep 2 from Fisher
(whatever), but other paraffin work as long as infiltration is
adequate. We use high profile blades for sectioning and less chance of
chatter, less stability we experience with bone microtomy using low profile
blades. Orientation will be important depending on what they want. A good
soak after trimming will help, often with RT water, then go to ice water
but don't oversoak. Plus charge slides work well, drain after pickup, and
dry sections FLAT at 37 to 40C overnight, although we find several days
works better to retain sections on slides.
Good luck
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
At 04:21 PM 9/13/2007, you wrote:
>Does anybody have a protocol for decalcification and processing of shrew
>mandibles?
>They are just a little over a centimeter in length and not very thick so I
>don't think the decaling would be very long.
>Haven't done this type of tissue before so I'm wondering if anybody out
>there has some hints for sectioning. The person who brought them said they
>could be hard to cut.
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
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