Dear Pat,

I do mostly fluorescent staining and I really like the results I get with a Hoechst counter-stain.  This stains the nuclei of all normal cells a light blue which is visible through a DAPI filter.  It provides nice contrast with the typical fluorescent visualization signals used.

The procedure I use is very simple.  

1)Make up a stock solution of 10mg/ml bis-benzimide.  (keep refrigerated for future use)
2)Use this stock to make up a "working" solution.  Dilute bis-benzimide 1:12,000 in 1X PBS.
3)After you have completed your antibody staining, immerse your slides in the "working" solution for 8 min.
4)Wash slides in buffer and coverslip.

I usually keep refrigerated and reuse the "working" solution for a month.  Bis-benzimide is a carcinogenic so use proper precautions.

Hope this helps,
angeline
medical college of georgia
amartin@mail.mcg.edu





>Date: 13 Feb 2001 09:26:08 -0600
>From: "Patrick M. Haley " <pmhales@cybergap.net>
>Subject: fluorescence counter-stain

Fellow netters,
    I have a very novice understanding of the techniques used in this area,
and I  have a researcher who would like to know that is the counter-stain of
choice for immuno-fluorescence. I have spoken to some other techs, and they
do not use a counterstain.

Many thanks.

Pat
HTN, inc.