Re: [Histonet] Giemsa as counterstain

From:James L Burchette

Erin - here is the protocol I use for staining melanin with Giemsa.

Post DAB Giemsa Counterstain for Melanin

From DAB, wash slides in tap water.
Counterstain with hematoxylin, wash, “blue, and wash per usual technique.
Rinse with DI water.
Place slides in full strength May Grunewald solution for one minute.
Pour off MG solution and drain excess from slides.
Pour on working Giemsa solution for two minutes.
Gently rinse slides in tap water for 1 minute.

Place slides in 95% ETOH for 2 minutes. **
Transfer slides to 95% ETOH with colophonium resin for 2 minutes. **
Transfer slides to a second 95% ETOH with colophonium resin for 2 minutes. 
Dehydrate and clear slides, mount with permanent mounting media.

**gently agitate or dip slides to facilitate removal of excess stain**

Giemsa working solution:
        2.5 ml Giemsa stock in 50 ml DI water

Colophonium working solution:
        10 drops 10% colophonium solution in 100 ml 95% ETOH

Giemsa, Sigma GS-1L
May-Gruenwald, Sigma MG1-L

Note: Differentiation can be accomplished with a 0.1-0.2% acetic acid 
solution. This will be very rapid in comparison to the 95% ETOH with 
colophonium. It will also remove the hematoxylin. A hematoxylin 
counterstain is not necessary, but does make a nice combination with the 
Giemsa counter stain. 

Jim Burchette, HT(ASCP) QIHC
"A simple histotech from a little country hospital in North Carolina"

"Martin, Erin"  
Sent by:
04/24/2007 11:11 AM


[Histonet] Giemsa as counterstain

Hello all,
Have any of you every used giemsa as an IHC counterstain for melanin 
containing tissues?  It  apparently turns the melanin green which 
differentiates it from the DAB.  My docs have seen this and want to use it 
here.  I bought giemsa solution from American Master Tech and tried it, 
but the docs said it doesn't look right.  If you have used giemsa for this 
purpose or know what I am doing wrong, I would appreciate any input!
Thanks so much,
Erin Martin
San Francisco

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