bone histology/reply/new woven bone

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From:Gayle Callis <> (by way of histonet)
To:histonet <>
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One of the problems confronting labs doing decalcified bone is
dealing with the potential understaining of the bone components.

One thing to try is do not do a regular hematoxylin and eosin stain
procedure, by regular, I mean going through a differentiation step,
acid alcohol or acetic acid.  One trick is to overstain with the
hematoxylin a bit (for Gill type hematoxylins, try 10 minutes, or with
Harris's, same thing or a tidge longer) avoid the "destaining" step
altogether, blue the section and do one section with eosin/phloxine
staining (10 dips, to be sure and demonstrate osteoid) and on the other
section, no counterstain at all.  This will sometimes bring out some of the
newly laid down bone vs older lamellar bone.  Eosin tends to mask some of the
hematoxylin staining of lamellae, if you stain too long.  Richard Allan
hematoxylin 1 for 10 minutes also works (our stain).  What you are attempting
is overcoming the effects of acid decalcification on staining.

Also, try a toludine blue stain, which may be the biggest help of all,
just don't overstain with this stain either.  Age of animal, species,
type of decalcification (you did not say which) , endpoint determinations
faithfully done  ALL affect the staining.   Try a 1% toluidine blue, or even
0.5% aqueous solution, stain for 1 minute, rinse, air dry and coverslip.
You can increase the time if needed.

Sometimes tweaking the method a bit, improves how the bone components look.
Undecalcified sections would look better, but that is often not possible.
Surface staining of acid etched ground sections in methylmethacrylate would
be superior, but if you do not do this type of embedding, try some of the
other "tweaks"

Good luck
Gayle Callis

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