Re: [Histonet] tissue of the week - shrew mandibles

From:Gayle Callis

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
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 
>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
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610

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