Re: bone marrow decalcification

From:Gayle Callis

Good points, but remember stirring also mixes Ca (released from bone and
then tends to settle to container bottom) will be important IF you want to
do a chemical endpoint test.  The test aliquot  shoulded be pipetted
aliquot from bottom of that container where this Ca resides.  The only time
this may be a problem is near endpoint, when you are attempting to detect a
minute amount of calcium. 

Nothing wrong with stirring or other agitation (ie platform
rocking/rotator, bubbling air through solution) but agitiation during
decalcification is a somewhat debatable point. I preferred stirring when
decalcifying 25 rat knees in Shandon nylon bags suspended in a large
container of decalcifier (endpoint tested with FAXITRON, xray method), but
mainly to disperse CO2 bubbbles formed during decalcification.  The knees
tended to float to surface!   Suspending bone in the decalcifier probably
achieves same even removal of calcium, this permits any decalcifier access
to all sides/surfaces of bone.  

Obviously, the effect of acids on antigens will determine staining, and not
all antigens can survive the assault.  One should test decalcifier to
assess if the immunostaining will work, you may have to use EDTA or formic
acid may work, even dilute HCl???    

At 12:38 PM 3/3/02 -0500, you wrote:
>Hi HistoNetters
>Patsy Ruegg pointed out that formic acid has proven to be a good agent for
>decalcification with subsequent IHC.  I don't disagree.  In fact, more of my
>customers use some form of formic acid decalcifier than anything else.  I
>have just found, in my own empirical experience, that I have gotten superior
>results using EDTA.
>Patsy also asked if microwaving itself affects the results, and the answer
>is that it only speeds them up.  Decalcification speeds can be highly
>temperature dependent.
>Furthermore, the use of an effective stirrer can ensure that fresh
>decalcification fluid is always in contact with the specimen.
>best regards,
>Steven Slap
>Marketing Manager/Microwave Product Specialist
>Hacker Instruments & Industries, Inc.
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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