Re: H&E of decalcified bone samples

From:Gerard Spoelstra <>

Dave, do you moniter the decalcification process to find the endpoint,
because once you go beyond the endpoint is when you start to get lose of
nuclear staining. We use 5%Nitric acid for most of our decalcification  .
The Nitric acid is moderately rapid.Is the bone already cut to size when
you begin decacification? If you left it in 5% Nitric Acid for 5hrs then
change the solution. After 1/2 an hour check it by putting
about 5ml in a test tube to which you add conc ammonia solution to achieve
a just alkaline solution
with litmus paper.Any calcium coming out will turn the solution cloudy. You
continue with this until no more calcium comes out. Mouse bones should'nt
take long to decacify.
Gerard Spoelstra
Biomedical Science
Murdoch University
Western Australia
At 11:39 AM 1/03/01 -0500, you wrote:
>Hi histoneters,
>I was hoping someone could help me out with a staining question.  I process
>mouse tissues and am currently working with front and hind paws and joints
>to study inflammation.  I use Cal rite from Richard Allen and decalcify
>according to their recommendations.  Usually no more than 24-48 hours for
>hind limbs.  (This is after 4-5 days of fixation in NBF) I also perform all
>fixation and decal on a tissue shaker to help facilitate penetration of
>reagents.  I feel this is the minimum to have demineralized samples without
>jeopardizing staining quality.  However, there is more eosinophilic staining
>occurring thank undec'd samples from the same animals.  I know that this is
>a result of the acid decalcifying process(and we don't have time to use
>EDTA)   Is there any way of soaking the slides in a higher pH PBS to bring
>the samples to pre-decal Ph to improve nuclear staining?  Any other
>suggestions are appreciated.  The staining is throwing off the ability to
>interpret which cell types are in the tissue.
>Thanks in advance
>Dave Kinsley MS,HT/HTL(ASCP) 
>Schering-Plough Research Institute
>Department of Immunology 
>2015 Galloping Hill Road
>Kenilworth, New Jersey 07033
>Phone:  908-740-4669
>Fax:      908-740-3084

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