[Histonet] Alizarin Red S Staining Protocol

[Tighe,Sean T]

Good afternoon,

In the past I have been using a 40mM Alizarin Red S Solution from 
Millipore to stain my cell cultures but I am now seeking an alternative. 
I plan on preparing fresh Alizarin Red S by adding the powder to 100ml 
of distilled water. In reviewing the literature, I have come across many 
labs using 1% ARS or 2% ARS and I am not sure if this concentration 
significantly matters. Furthermore, I understand some people use 
McGee-Russell's procedure using a pH of ~4.2 whereas others use Dahl's 
procedure using a pH of ~6.3. However using a pH of 4.2 seems illogical 
to me as this could remove some calcium from the monolayer.

Currently I plan on fixing the cells with 4% Paraformaldehyde in PBS 
(pH 7) for 15 minutes, washing twice with water and then staining the 
cells with 2% ARS in water (pH 6.3). Do you see any problems with this? 
Also, should I stain for 5 minutes or roughly an hour with the ARS dye?

I have had some problems with extracting this dye in the past. The 10% 
acetic acid does not seem to remove all of the dye and the results are 
variable. Note: before I extract the cells with acetic acid I wash the 
cells four times with water.

Regards,
Sean Tighe