More on Osteoid staining on decalcified bone

From:Gayle Callis <>

Barry makes excellent points here.   Some hematoxylins stain bone with
different tinctoral qualitiy and are better (in my estimation, having
played with them) for decalcified bone embedded in paraffin. 

Ehrlichs hematoxylin is wonderful, cartilage is well stained, as are nuclei
(provided you have not overexposed the bone to acids during
decalcification! and with care on counterstaining with eosin/phloxine (in
our lab) or other eosin formulations (per Barry's suggestions) osteoid is

Harris hematoxylin works well often better without the acid alcohol
differentiation step if bone is over"decalcified".  My best results have
always been with the classic Harris hematoxylin using mercuric oxide, the
forbidden chemical these days.   With sodium iodate as the oxidizer,
Harris's was weaker.  Surgipath makes a good Harris, and if this is fresh,
worth a try.  My inhouse Harris oxidized with sodium iodate was always a

Mayers never gave me the depth of staining I needed for decalcified bone,
and it took too long to do, so Gill II or III,  Richard Allans hematoxylin
1 plus some other formulations (commercial!) sometimes with extended
staining times, up to 10 minutes to help counter the effects of acid
decalcifiers.  One can never totally escape the effect of acids on nuclei
and other proteins, if that is a problem, EDTA is an option.  

It is important to note that before going into alcoholic eosin OR
eosin/Phloxine, one must carefully rinse bluing reagents away adequately, a
minimum of 1 minute (we do 2 min) to remove the alkaline bluing chemicals.
The pH for eosin staining is as critical for this counterstain as correct
pH is for hematoxylin staining, and without good rinsing, you will have
some funky, poor eosin staining.  This is also a factor with soft tissue
staining. Also, care in changing the 95% alcohols after the eosin steps was
frequent, to prevent overstaining with the eosin.  If your bone is red,
red, red, you need to back off the eosin.  

Good H&E staining on decalcified bone requires a bit of work, rethinking
what you do a bit, and then playing with some stains, times, etc in order
to see bone morphology.  I have some bone samples where lamellar structure
are obvious as is osteoid, ruffled borders on osteoclasts, and so on.  

Enough, happy staining to all- and Barry, Culling would be smiling at your
eosin staining, he loved aqueous eosin solutions and their wonderful range
of colors. 

Gayle Callis

At 02:17 PM 8/1/01 -0500, you wrote:
>I agree with Gayle re osteoid staining. You can demonstrate osteoid
>using  a good H and E.  There must however be hundreds of opinions
>regarding  what is a good H and E.
>I think that we have to accept the fact that  H and E can be used in
>different ways to demonstrate different histologic features to their
>best advantage. A weak eosin will often still allow eosinophils to be
>easily located and identified.
>I think that one of the problems that I have encountered is the eosin
>counterstain. Here in the States the most common method seems to be to
>use alcoholic eosin and an alcohol differentiation.  The technique that
>I was taught (admittedly in the middle ages) was to use an aqueous
>solution containing eosin Y and B. After staining there was an initial
>differentiation in tap water and a rapid dehydration. In my opinion this
>produces a counterstain with a broader range of shades of the eosin than
>can be achieved using alcoholic differentiation.  Having said that, to
>show the osteoid to its best advantage as a clear pale pink, you will
>have to accept a weak eosin sta

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