RE: [Histonet] Alizarin S mindbender question

From:"Rittman, Barry R"

Hi Julien
Greetings from Houston where it is never cold. Come visit us sometime if you want to escape from  the north.

I have not worked with fish apart from ingesting several; however I have used an alizarin/ alcian blue technique several times to stain bone and cartilage for frogs and rodents.
The technique that you use removes soft tissues and I am unclear as to why this is necessary. The techniques that I have used have retained the soft tissue and have made them permeable to clearing agents so that the details of bone and cartilage are very clear.
My first thought on your technique is that the KOH is made up too strongly by mistake, can easily happen or that there is still trypsin present and acting (although the usual pH range for trypsin is 7 to 9). Both would explain the soft tissue maceration.
I am not sure how long you use the trypsin or its concentration although it generally is fast acting and it is usually only active for about 3 hours. Trypsin can be sold with some gelatin in it as a stabilizing agent but not sure if this is the case here.
The cloudiness may be a reaction between remnants of trypsin and the alizarin.
Just some thoughts, not sure if these are correct or just BS.
Good luck

-----Original Message-----
From: [] On Behalf Of Julien Lambrey de Souza
Sent: Wednesday, June 08, 2005 3:30 PM
Subject: [Histonet] Alizarin S mindbender question

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 -20īC freezer. The freezer has had a problem and 
freezed to -40īC 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.

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