[Histonet] Counterstain for dual chromogenic

[Tony Henwood]

Try ethyl green:
1.	0.1N Acetic Acid
	Add 6ml of glacial acetic acid to 1000 ml of distilled water.  
2.	0.1N Sodium Acetate
Add 4.102 g of sodium acetate to 500 ml of distilled water
3.	pH 4.2 buffer
Add 755 ml of 0.1N acetic acid to 264 ml of 0.1N Sodium Acetate and adjust the pH to 4.2 with 1M NaOH
4.	Ethyl Green Solution
To 100ml of buffer add 2g Ethyl Green (CI 42590)
Can be stored at room temp.  
If staining turns bluish than the solution has started to deteriorate.

Stain for 5 minutes. Do not rinse in water for too long, it tends to extract the green staining

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Eva Permaul
Sent: Wednesday, 13 April 2011 1:37 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Counterstain for dual chromogenic

Good morning,
I have been attempting to do some dual chromogenic staining. I am using a mouse and a rabbit antibody. For the mouse antibody I am using an HRP-mouse secondary followed by AEC for visualization. For the rabbit antibody I am using a biotinylated rabbit secondary, followed by ABC-AP and vector blue. My problem is what to use as a counterstain. From everything I have read so far there isn't one that would work without having some problems. I was thinking of trying a very light Hematoxylin and see if it doesn't disrupt the vector blue visualization too much. 
Does anyone else have any suggestions?
Thanks,
Eva

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