[Histonet] RE: Toluidine blue background

[Thompson, Samantha J]

We use T blue for frozen sections that we make using Toludine Blue O powder (ITEM SPT1182) from American Master Tech. Disolve 2.5 g of the powder into 500 mL DI H2O. I warm the water to help the powder completely dissolve and continually stir the until solution is made. I usually make up in a gallon size container and just pour into coplin jar for use. I fix the frozen sections in 100% ETOH ~30sec and rinse well in running tap water ~15 - 30 secs. Stain in T-Blue for 3 minutes and then set the stain with a borax solution* for ~ 15 secs. Rinse in running tap water 10 sec. and airdry. Clear with xylene or substitute and coverslip. I never have any background staining and no issues with remaining OCT. 

The T-blue lasts for about a month depending on how many slides you are running through it. I use the borax for two days before dumping and refilling. 

*borax solution made....100mL borax + 3000mL DI H2O. Again I make this up in a gallon size to pour as needed

I hope this helps!

Samantha Thompson
Pathology Manager
Dawes Fretzin Dermatology Group


________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] on behalf of O'neil, Beth [oneilb <@t> wvuhealthcare.com]
Sent: Tuesday, December 16, 2014 9:36 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Toluidine blue background

We would like to use Toluidine Blue for staining frozen sections of kidney biopsy.  We finally are able to get the stain dark enough, but we can't get rid of the background caused by the OCT.    We've washed with water, alcohols, and PBS  with no success.  Is anyone out there using Toluidine Blue for frozen staining?  If so, what is your recipe and how do you get rid of the background?
Thanks

Beth Ann O'Neil, MT(ASCP)SC, HTL, QIHC
oneilb <@t> wvuhealthcare.com<mailto:oneilb <@t> wvuhealthcare.com>
Histology Supervisor, Technical Specialist
Lab:  304 - 293 - 6014
Office:  304 - 293 - 7629



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