From section VIII.a.vii. of the 2010 dissertation of Sarah Kigamwa Amugongo (pp. 34-36), which can be found at http://escholarship.org/uc/item/7dh1h9bz

Von Kossa Method with McNeal’s Tetrachrome Counterstain

Reagents:

1. Silver nitrate solution (5%) (Binds to calcium and phosphorus minerals)

Silver Nitrate 5 gm / 25 gm

Distilled water 100 ml / 500m

• This solution was filtered before each use and a dedicated silver funnel was used

since silver stains everything black.

• The silver nitrate was then poured into a dedicated staining container and covered.

• The solution was kept in a dark cabinet in light-proof container and was re-used.

2. Sodium Carbonate-Formaldehyde Solution:

(acts as a developer by turning Silver nitrate black).

Sodium Carbonate 5gm / 30 gm

Formaldehyde 25 ml / 150 ml

Distilled Water 75 ml / 450 ml

This solution was filtered before use.

3. Farmer's Diminisher – a fresh batch (210 ml) was mixed for each run

(This is a fixative that prevents silver nitrate from coming out).

Sodium Thiosulfate 20 gm

Potassium Ferricyanide I gm

Distilled Water 210 ml

• Once the potassium ferricyanide was added, this solution was stable for only about 45 min.

• The sodium thiosuflate was dissolved in the distilled water first, then potassium

ferricyanide added.

4. McNeal’s Tetrachrome Solution (2%) (Polysciences, Cat. #2783);

McNeal’s Tetrachrome* 2.0 gm /10 gm

Double distilled water 100 ml / 500 ml

• The dye was added and slowly brought to boiling point.

• The heat was turned off and the solution allowed to stir overnight.

• The solution was then filtered before use.

Staining procedure

Covering film was removed and sections de-plasticized in acetone (Kacena et al., 2004).

This was followed by rehydration through graded series of ethanol (100% EtOH, 95% EtOH,

70% EtOH and distilled water x 2). Staining was carried out as follows:

1. Sections stained in Silver Nitrate solution for 10 minutes in the dark

2. Rinsed in distilled water x 3 for 1 minute each

3. Stained in Sodium Carbonate-Formaldehyde solution for 2 minute

4. Rinsed in distilled water x 2 for 1 minute each (Potassium Ferricyanide to Farmer's added

at this point)

5. Stained in Farmer's diminisher for 30 seconds (timing was very critical for this step).

6. Washed in running tap water for 20 minutes

7. Rinsed in distilled water for 1 minute

8. Stained in McNeal’s tetrachrome solution for 10 – 15 minutes

9. Rinsed in distilled water x3 for 1 minute each

10. Dehydrated in 1 change of 70% EtOH, 95% EtOH and 100% EtOH; blotted between

changes of alcohol

11. Cleared in 2 changes of xylene

12. Coverslipped with Eukitt

13. Allowed to dry under the fume hood overnight.

According to the author, this method gives good results. I hope this helps!

For some suggested alternatives you might consider having a quick read of https://www.researchgate.net/post/What_staining_method_would_you_recommend_for_distinguishing_between_osteoid_lamellar_and_woven_bone_on_FFPE_decalcified_bones

Hi Barae, thanks for your suggestion. I however have material which is already decalcified and this protocol is using undecalcified material. I am not sure if this protocol is therefore still valid.

Which tissue components do you want to stain on one slide?

I like to see the difference between primair and secundair bone.