RE: [Histonet] Alizarin S mindbender question

From:Shirley Powell


I have performed the Dawson's technique (page 549 in CFA Culling third
edition) on human embryos years ago using Alizarin to stain bone.  I am not
sure if this is suitable for what you are doing but if you contact me I will
be happy to talk to you about what I encountered in the procedure.  Your
email address did not show up in the message.  My email address is or you can call me.

Shirley Powell
Mercer University School of Medicine
Macon, GA 
-----Original Message-----
[] On Behalf Of Rittman,
Barry R
Sent: Friday, June 17, 2005 8:46 AM
To: histonet
Subject: RE: [Histonet] Alizarin S mindbender question

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
Just some thoughts, not sure if these are correct or just BS.
Good luck
-----Original Message-----
[] On Behalf Of Julien
Lambrey de Souza
Sent: Wednesday, June 08, 2005 3:30 PM
To: [Histonet] Alizarin S mindbender question

=0AHello 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=0Dfirst 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:
=0A1) dehydration in EtOH ---- the fish turn out fine
2) stain in alcian blue solution (EtOH+acetic acid+alcain blue 8GN)---- fish=0Dturn 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
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=0Dcurl 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

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=0Dhave 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,=0DEvolutionary biology,
UQAR, Quebec.

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