[Histonet] Von Kossa stain

[Brunella Spaggiari]

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 (see attachment), 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