[Histonet] Re: DAB and Nuclear staining

[Michelle MacVeigh-Aloni]

Hi Melissa,

We use Mayer's hematoxylin for 5 min and then rinse in tap water for at least 5 min (to blue the stain). 
If your DAB stain is weak, you can counterstain for even shorter time. It works great!

The other option would be 1% Methil Green in 0.1M Sodium Acetate. My Methil powder is extremely old and probably this is the reason the stain is very weak, but it makes great contrast with the brown of the DAB.

Good luck
Michelle 



----- Original Message ----- 
From: "Melissa Mazan" <melissa.mazan <@t> tufts.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Thursday, September 03, 2009 6:18 AM
Subject: [Histonet] DAB and nuclear staining


> Hi all,
> I have been having a problem with counterstaining for nuclei - I'm 
> looking at mouse lung tissues and staining macrophages with F4/80 (rat 
> anti-mouse). I use a negative serum control and a rat IgG isotype 
> control. I see very clear staining of macrophages - but when I 
> counterstain with hematoxylin or nuclear red, everything becomes muddy 
> and I can no longer discern the DAB staining. Can anyone offer advice? I 
> am using the Vector hematoxylin, and have tried as little as 5 seconds. 
> Melissa