Many years ago, I also did some work with MMA sections of undecalcified 
bone. I encountered much the same problems that you are experiencing.=20
So, a couple of suggestions:

To make the mineralized portions of the bone black (rather than brown), 
treat the sections with silver nitrate as in the regular von Kossa=20
technique, wash well in distilled water and then treat the sections with 
0.5% aqueous Hydroquinone. This is a good reducing agent and will=20
convert the silver to a stable black metallic form. The section must be 
well washed before this step or you will get all kinds of ugly 
precipitates. If you have washed really well, you can usually omit the 
thiosulphate step as all it does is to reduce the contrast of the silver.

Once you have good dense black calcium deposits , you can counterstain 
with a van Gieson (picric acid and acid fuchsin). This will give 
beautiful bright red osteoid seams, and most of the bone marrow will be 
yellow. You may have to play around with the timing of the van Gieson to 
get optimal results. But both dyes are quite small molecules and should 
penetrate quite fast; I would start at around 2 minutes and see how it 
looks. Slow dehydration will also remove a lot of the red and yellow, so 
don't spend too long in the dehydrating alcohols.

Paul Bradbury
Kamloops, BC
Canada


Brunella Spaggiari wrote:

>Hello everybody,
>
>we work on undelcalcified bone tissue (human, rabbit) fixed in PFA 4% and embedded in methylmethacrilate (MMA).
>>From our samples we obtain 50 µm thick sections which can contain titanium implants.
>We tried to perform Von Kossa stain, always on undeplasticized sections, but we met several problems:
>
>- calcium deposits after silver nitrate+UV light did not appear black but brown, and, after sodium thiosulphate, brown stain turns more and more light;
>
>- we could not find a suitable counterstain for osteoid: nuclear fast red and neutral red do not stain at all, basic or acid fuchsin stain too much and cover the underlying brown..
>
>Some suggestions? Are there other good histological stainings for undeplasticized bone sections in your experience?
>
>Thank you!
>Brunella
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>
>  
>


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