RE: intracellular staining of serotonin

Stacy Powell wrote:
>...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 [previous] 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.


Flow for intracellular antigens is usually associated with higher 
background fluorescence than is typically encountered when analyzing 
surface markers. Is your primary antibody polyclonal or monoclonal? 
You will almost certainly have higher background with a polyclonal 
antibody, and, as a rule, higher background with indirect 
immunofluorescent staining than with direct staining. Also, 
fluorescein signals tend to be in the same spectral region as most 
cellular autofluorescence. But the most important thing is: How many 
serotonin receptors do you expect there to be in each positive cell? 
If you're looking for a hundred or so receptors, the variable 
background from all the factors I just mentioned will swamp the 
difference in fluorescence signal due to different numbers of 
receptors in the positive and negative cells.

-Howard