[Histonet] Alizarin Red Troubleshooting

[Amos Brooks]

Hi,
     I seldom do this, but I'll bet I know EXACTLY what the problem is. Try
it again and stop the rehydration at 95% ETOH then put it directly into the
Alizarin Red (double check the pH of course). This will surely fix it. The
calcium gets rinsed out when it is rehydrated. The same thing happens with
Von Kossa. I also try to abbreviate rinsing after when dehydrating to
coverslipping.

Let us know if you still have trouble,
Amos


Message: 18
Date: Thu, 19 May 2011 09:30:27 -0400
From: "Phipps, Amanda" <Amanda.Phipps <@t> nationwidechildrens.org>
Subject: [Histonet] Alizarin Red Troubleshooting
To: "'histonet <@t> lists.utsouthwestern.edu'"
       <histonet <@t> lists.utsouthwestern.edu>
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20CE7246011C12489BA04BEB85CCDA1F0351BF252B <@t> res2k7ms01>
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Hi All

We are currently trying to stain for calcium deposits using Alizarin Red.  A
few months ago our protocol worked fine, but this time we are getting zero
staining.  Reagent has been made fresh, and pH is between 4.1 and 4.2.

Here is our protocol.

Deparaffinize to distilled H20
Stain slides with Alizarin Red S for 30 seconds to 5 minutes and observe
reaction microscopically
Shake off excess dye and blot sections
Dehydrate in acetone 10 seconds to 20 seconds, then acetone-xylene (1:1)
solution 10-20 seconds
Clear in xylene and mount in synthetic mounting medium.

Any help is greatly appreciated!!


Amanda Phipps, HTL (ASCP)
Histotechnologist
Morphology Core Lab
The Research Institute at Nationwide Children's Hospital
700 Children's Drive
Columbus, Ohio 43205
(614) 355-3474