This is the method I used for cell cultures on wafers.

VON KOSSA FOR CALCIUM

1)   Wash wafer with PBS or medium without FCS, warm.
2)   Fix with 10% buffered formalin phosphate for 2 hours to  overnight, RT.
3)   Wash with distilled water at least 5 times.  Let last two rinses sit in
water     for 5     minutes.
4)   Place 5% AgNO3 (made fresh, 5g/100ml) onto wafer and place under light
for     6 10     minutes. (Normally 10 minutes), until nodules are black.
5)   Wash with distilled water >5 times (leave water on for a few minutes
for 2 of     the     washings).
6)   Differentiate in 5% hypo (sodium thiosulphate) for 2 minutes.
7)   Wash with distilled water, 3 times.
8)   Stain with toluidine blue ( diluted 1:5) for 10 seconds.
9)   Wash several times with distilled water.
10)  May mount (cell side up) once dry, if desired.


Hope this helps.

Lesley Weston.



on 17/01/2003 2:37 PM, Mandy Annis at mandy_annis@usgs.gov wrote:

> A co-worker is in need of the recipe for Von Kossa staining method for
> bone.  A search of our histo recipe books yeilded widely differing recipes.
> Any and all recipes that you would be willing to share would be
> appreciated!
> 
> Thank you,
> 
> Mandy Annis
> 
> **********************************************************************
> Mandy L. Annis
> Columbia Environmental Research Center
> 4200 New Haven RD  Columbia, MO  65201
> 
> Phone:(573)-441-2940
> Fax:(573)-876-1896
> e-mail:mandy_annis@usgs.gov
> 
> 
>