[Histonet] Alizarin S mindbender question

[Julien Lambrey de Souza]

Hello histonetters,

In our lab we do routine staining of fish specimens by the Clear and 
stain technique of Dingerkus and Uhler 1977, and some of the existing 
modifications with very nice results.

But in the last few days, this technique has turned sour....

Something is happening to the fish when they are left in the Alizarin 
red S solution made up with 0.5% KOH. The solution has been made fresh 
several times but gives the same result each time. The fish seem to 
contract (muscular?) so much that the caudal becomes deformed (S 
shaped), the fin rays curl up and detach and the cranial elements also 
seem very fragile. My first thought was that the KOH solution was too 
harsh, but then I realized that the same KOH (0,5%) is used with these 
fish in the bleaching solution without harming the fish (apart from 
normal bleaching).

The protocol is summarized as follows:
1) dehydration in EtOH ---- the fish turn out fine
2) stain in alcian blue solution (EtOH+acetic acid+alcain blue 8GN)---- 
fish turn out fine
3) neutralization in borax ---- fish are still fine
4) bleach in H2O2 solution (H2O2 3%+ KOH 0,5%) ---- fish still very nice
5) trypsin digestion (borax+H2O+tryspin) ------ although trypsin has 
cleared the specimen of its "meat" the fish is still very nice and fin 
rays are intact.
6) Alizarin red S solution (KOH 0,5%+alizarin S to turn solution deep 
purple) ----- almost on contact with the solution, the fish rinkles, 
rays curl up and detach... the specimen becomes useless. (The solution 
used to color the bones red and the specimen was then cleared of excess 
red by KOH bath).

I have tried changing borax and KOH solutions with no improvement. 
Could it be that Alizarin S has gone bad? Is this even possible? We 
have had very high temperatures in the lab lately (30 degrees celsius), 
could this have influenced the solutions? I am puzzled. Also, we keep 
our trypsin in the -20´C freezer. The freezer has had a problem and 
freezed to -40´C for a day. Could this have altered the trypsin in a 
way that specimen exposition to this trypsin is incompatible with a 
later exposition to alizarin? (I am out of good ideas, so I'm trying 
absurd ideas....).

If anyone is willing to take a shot, I will accept all suggestions 
because I am high and dry.

Thanks for any help.

Julien De souza,
Evolutionary biology,
UQAR, Quebec.