[Histonet] How to re-stain old specimen?

[Julien Lambrey De Souza]

Hello to all the histonet community,

We are doing the clear and double staining technique by Dinkerkus and Uhler (1977). For those who are not familiar with this technique, it consists in staining specimen with Alcian blue to reveal cartilage and with Alizarin Red to reveal bone, on whole fish that have been bleached (in H2O2 + KOH to remove chromatophores) and digested in trypsin (to make the fish transparent). The fish are then passed through a gradient series of KOH-Glycerin gradients to 100% Glycerin for storage (a few crystals of thymol are added to avoid mould formation). The technique works wonderfully well. 

But here is the catch. The problem is that the red Alizarin stain tends to fade with time and therefore all observations are done within 5 days of staining. In my particular case here, I have 2 old specimen (more than a year old) that have lost their red coloration but I have to retrieve bone information from them and I have no way of getting equivalent specimen to redo the whole staining procedure. So I have to re-stain these 2 fishes, go backwards in the protocol towards the red staining.

According to the original protocol, I stain my samples in a KOH 0.5% solution of Alizarin red for a day. Then to remove excess red, I go through the above mentioned gradient series of KOH-Gylcerin towards the 100% glycerin for storage.

I was thinking of reversing the gradient series in order to go back to KOH0.5% and Alizarin red, but in what proportions and where do I start incorporating the red stain? This is what i'm figuring:

step 1: 100% Glycerin
step 2: 75% Glycerin + 25% KOH(0.5%)
step 3: 50% glycerin + 50% Alizarin red KOH(0.5%) solution
step 4: 25% Glycerin + 75% red solution 
step 5: 100% red solution.

Then I would gradually go back up to Glycerin 100%. But I'm wondering if this would be to much KOH for the specimen.

I only have one shot at this, these 2 fish are valuable. This is why I'm looking for input on the subject (and where better than the histonet to find this). Any thoughts are very welcome.

Thanks,

Julien Lambrey de Souza
Biologie Évolutive,
Université du Québec à Rimouski,

(418) 723-1986 #1438