RE: [Histonet] Mineral deposits and von Kossa & alizarin S

From:"Kemlo Rogerson"

We recently found what appears to be mineral within

mouse heart tissue.  This mineral stains with von

Kossa, but was negative with Alizarin Red S, with the

exception of a single mineral deposit in one animal. 

The hearts were fixed in neutral buffered formalin

(NBF).  Is it possible that the calcium leached out in

the NBF and that is the reason for the negative result

with Alizarin Red?  Some of the older literature

suggests using alcohol fixatives, but more recent

literature recommends using NBF.   If the calcium

leached out would you still expect to see non-staining

mineral deposits, or should the entire deposit be

dissolved away and absent from the tissue section?



I am not familiar with the Alizarin Red stain for bone and thought it
was used to demonstrate sites undergoing mineralization. For example
madder (a herb containing Alizarin) fed to cattle coloured their bones.
There is a technique for using Alizarin Red S to colour the mineralised
bones of fetal or newborn rats involving maceration with KOH and
Alizarin Red over many days. So I thought you used it specifically to
demonstrate developing bone.


Von Kossa's, on the other hand, like Lillie's method of silver
impregnation, demonstrates phosphates rather than calcium salts, but as
soluble phosphates are washed out it is essentially calcium phosphate
that is demonstrated. There are a variety of techniques available to
demonstrate calcium (phosphate) and Lillie proposes a block technique
after formol fixation;


1)    Immerse blocks in 6 changes of dist water for 20 min each.

2)    Place blocks in 1.7% (0.1M) silver nitrate and keep in dark.

3)    Remove blocks between 5 days and 10 days (experiment).

4)    Wash in 6 changes of dist water for 20 min each.

5)    Transfer to 2g sodium bromide in 94 ml dist water for 3 hours.

6)    Add 6 ml glacial acetic acid. 

7)    This mixture (2% sodium bromide- 6 % glacial acetic acid
decalcifying fluid is changed daily. 1 ml discarded fluid is tested
daily with 1 ml 2% sodium oxalate until there is no turbidity.

8)    You can the process the block, cut sections, stain with collagen
or elastin stains.


Calcified areas are black.


Brought to you from the disciple of that great man Prof R.D. Lillie MD.


PS Alcohol fixation is 'said' to be better. 




Kemlo Rogerson

Pathology Manager

Ext  3311

DD   01934 647057

Mob 07749 754194

Pager 07659 597107



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