RE: intracellular staining of serotonin

Well, we are analyzing the receptors for serotonin, not actually the
serotonin molecule itself. I realized I should have made that
distinction after I sent the email. 
The method we used previously, which was unsuccessful, we used LTK cells
which are neg. for the receptors and LZD7 cells which have been
transfected to have the serotonin receptor, but they showed up as
negative.

We permeabilized with 95%EtOH 4C 30 min
Fixed with 2% paraformaldehyde in PBS 30 min 4C
Incubated with Guinea Pig anti 5HT1a in Pharmingen Stain Buffer + 1%
normal goat serum overnight at 4C
Washed 3X
Added Goat anti Guinea Pig Ig -FITC 1hour 4C
Washed 3X
Analyzed by Flow

Do you have any thoughts on our method that may have given us a problem
as to why the LZD7 cell are showing up negative? Your thoughts would be
greatly appreciated.

Stacy

-----Original Message-----
From: Prussin, Calman (NIH/NIAID) [E] [mailto:CPRUSSIN@niaid.nih.gov] 
Sent: Monday, December 19, 2005 4:58 PM
To: cyto-inbox
Subject: RE: intracellular staining of serotonin

Stacy,

I don't think this is technically possible using present day technology.
All of the fix and perm protocols rely on fixation to immobilize the
(usually protein) antigen. Serotonin is a small molecule (176 Daltons)
and thus would be unlikely to be immobilized by current fixation
techniques, which typically rely either on cross-linking or denaturing
proteins to immobilize. As soon as the cells are permeabilized, the
serotonin would leak out.

Sorry.....

Calman
> _______________________
> Calman Prussin
> Laboratory of Allergic Diseases
> National Institute of Allergy and Infectious Diseases/ NIH
> 
> ----------
> From: 	Powell, Stacy
> Sent: 	Friday, December 16, 2005 12:45 PM
> To:	Cytometry Mailing List
> Subject:	intracellular staining of serotonin
> 
> Hello all!
> 
>  
> 
> Does anyone have any experience doing intracellular staining for
analyzing serotonin by flow? If so, what method of permeabilization do
you use?
> 
>  
> 
> Thanks!
> 
>  
> 
> Stacy Powell
> 
> Biologist
> 
> Southern Research Institute
> 
> 2000 Ninth Avenue South
> 
> Birmingham, AL  35205
> 
> 205-581-2243
> 
> powell@sri.org
> 
>  
> 
>