Do you want to know if a double staining is possible? or what a double staining implies? The answer to the first question: yes. To the second:

Anillin blue in plants stains cellose structures and alizarin basically stains calcium 

Miller JH, Kotenko JL. The use of alizarin red S to detect and localize calcium in gametophyte cells of ferns. Stain Technol. 1987 Jul;62(4):237-45.

A co-staining (double-staining) in the same structure would indicate the presence of calcium in the cellulose ("salty plant?") 

Thanks Janine :-)

As I understand, ARS only stains mineralized calcium (salts) and not ionic Ca2+.(?)

I use aniline blue to localize callose responses. In the co-staining experiment I saw strong deposits of ARS that I did not see in the ARS treatment alone. I was just concerned that ARS would be binding to aniline blue. However, now I have realized that the strong deposits I saw in co-staining were due to the pH of the aniline blue solution (pH 9) and not due to aniline blue itself.  In the co-staining experiments, I stained using aniline blue pH 9 first (overnight). After a brief wash in water, I  proceeded to ARS pH 4. I could not see the same intensity of ARS stain in individual  stains that were visible in co-stains. However, after I posted here, I learned that ARS solutions with pH ranging from 9 to 12 stain calcium deposits more intensively and with less artifacts:

Puchtler H, Meloan SN, Terry MS (1969) On the history and mechanism of alizarin and alizarin red S stains for calcium. J Histochem Cytochem 17: 110–124 

The  protocol I have suggests an acidic pH of 4,1. However, when I stained my samples using ARS pH9, there was a strong intensity in the structures resembling the same stain I saw during the co-staining with aniline blue. I, therefore, concluded that the overnight treatment in pH 9 increased the pH of the original ARS 4.1, conferring the observed strength in stain, probably because of insufficient washing between the two steps.  

I am however not completely certain about how ARS exclusively interacts with calcium salts and not Ca2+. Do you have any idea how?

Hi Geziel,

alizarin red S reacts with calcium cation to form a chelate - see for example: Wang, Y. H., Liu, Y., Maye, P., & Rowe, D. W. (2006). Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes. Biotechnology progress, 22(6), 1697-1701.

If you have a  strong stainings in areas you don't have when you use ARS alone it might be that the washing step is not sufficient? ARS should not stain / interact with Aniline blue as this has no Ca2+ anywhere. 

Have you tried the staining with red before blue (as an unusual control to see if you have more blue in areas it shouldn't be when red is used in the first step)?

Janine

Thanks for the very informative discussion Janine. 

In my previous comment I mentioned that ARS does not stain Ca2+ cations.  I was implying that my fixation procedure (which includes incubation in aqueous solutions of 70% ethanol) washes off ions from the sample? This is my general assumption based on the fact that incubation in acidic pHs remove immobilized 'stainable' sites from mineralized samples [Puchtler H, Meloan SN, Terry MS (1969) On the history and mechanism of alizarin and alizarin red S stains for calcium. J Histochem Cytochem 17: 110–124]. In this paper they also show that ARS precipitates non-specifically with a LOT of other salts... (scary!!!).

In [Moester MJC, Schoeman M a E, Oudshoorn I, van Beusekom MM, Mol IM, Kaijzel EL, Löwik CWGM, de Rooij KE (2013) Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone. Biochem Biophys Res Commun 443: 80–85] they state that '(ARS) displayed specificity for mineralized nodules' even though they cite Puchtler et al. (1969) where they show the non-specificity of ARS.

In any case, I am assuming that it is generally accepted (although controversial...) that ARS will bind specifically to immobilized calcium in samples that were previously fixed in aqueous solutions of neutral pH? I guess there is no way to be completely certain that ARS precipitation truly represents the presence of mineralized  vs immobilized calcium in these situations?  I believe I can say that those regions represent 'immobilized or insoluble' calcium deposits...? 

I say this because in vivo calcium staining techniques (as in Moester et al. (2013)) can detect halos of fluorescent that are not seen in fixed ARS samples, suggesting the presence of soluble ionic Ca2+ that is washed off during fixation. 

My concern is just not to conclude erroneously about what I see in the stainings.  :-/

Hi, that' s really interesting  ...  It seems to be much more controversial than I was aware of. Your statement "I can say that those regions represent 'immobilized or insoluble' calcium deposits" is safe, but I think we/you still don't have an explanation for the additional staining of ARS when combining with aniline blue. - I will think a bit more about it and when I get an idea I will for sure contact you.