RE: ki-67 stain permeabilization?

Hi Alessandro,

I suggest you to use a kit for intracellular cytokine staining such as eBioscience
Fix/Perm (either for cytokine or for FoxP3), BD Cytofix /Cytoperm or similar. They both
work very well. Generally, you do surface staining, then wash, fix, wash with
permeabilizing medium and incubate with Ki-67 Ab in perm medium again. If you don't want
to use this buffers, you can fix cells in 1% PFA for 10 min, then wash a couple of times
with PBS + 0.02% tween 20 and resuspend cells in the same medium together with Ki-67 Ab.
This last protocol worked for me a couple of years ago but take into account that the
fluorescence of your surface antibodies will be much more affected compared to commercial
kits. Last thing, I don't know if you are studying human lymphocytes, but be sure to
acquire a sufficient number of events (>100.00, let's say), since Ki-67 is poorly
expressed on peripheral lymphocytes.

Enrico

-- 
Enrico Lugli, PhD
Research Fellow, ImmunoTechnology Section
Vaccine Research Center, NIAID, NIH
Bethesda, MD 20892-3015
Phone : (301) 594-8602
Fax   : (301) 480-2788
________________________________
> Subject: ki-67 stain permeabilization?
> Date: Tue, 1 Jul 2008 13:09:11 +0200
> From: serra@drfz.de
> To: cytometry@flowcyt.cyto.purdue.edu
> 
> 
> Dear All
> 
> 
> 
> Can anybody suggest which is the best method to permeabilize cells in order to obtain
the most reliable staining with Ki-67 Ab from BD?
> 
> Thank you very much
> 
> 
> 
> Dr. Alessandro Serra
> 
> Cell Biology Group
> 
> Deutsches Rheumaforschungszentrum (DRFZ) Berlin
> 
> Charitéplatz 1
> 
> 10117 Berlin
> 
> 
> 
> Phone: +49-30-28460 765
> 
> Fax: +49-30-28460 603
> 
> Email: serra@drfz.de

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