AW: Question about Introcellular antibody staining

Hi Yang,

I don't know which cytometer you are using but setting the FSC threshold to zero should
be avoided. At 0 you only get noise signals. Increase the threshold and if necessary the
gain of the FSC to see where your cells are in the FSC/SSC scatter plot. If you can't get
any signals then you have either lost your cells or the cells are totally fragmented by
the fix and perm procedure. It makes no sence to use a viability marker like 7-AAD or PI
when you are fixing your cells because by that you are killing your cells for
intracellular antigen staining. It's only useful as a quality control of your
permeabilization procedure i. e. are all cells permeabilized or not. Permeabilization and
fixing procedures should be as gentle as possible. FSC and SSC signals should not change
dramatically compared to unfixed cells. If this is the case then there is something wrong
with the protocol. Protocols always have to be adapted to the cell type in use. Antibody
incubation for an hour seems to be very long. If the antibody hasn't bind in 20 min it
will never bind. Also the incubation at RT is contoversal discussed. 
But first of all you have to optimize the fix and perm protocol to get a nice detactable
cell population.

Good luck

Volker

Volker Eckstein PhD
Dept. of Internal Medicine V
Medical School of the University
University of Heidelberg
Im Neuenheimer Feld 410
69120 Heidelberg
GERMANY
volker_eckstein@med.uni-heidelberg.de
Tel. +49 6221 56 37356
FAX: +49 6221 56 5775



-----Ursprüngliche Nachricht-----
Von: Yang WANG [mailto:doll731@mail.ku.edu] 
Gesendet: Montag, 14. November 2005 00:42
An: Cytometry Mailing List
Betreff: Question about Introcellular antibody staining


Hi everyone,

I am staining embryonic stem cells using different introcellular antibodies as 
differentiation marker, however, they all showed positive for over 95% , which 
can't be true. When I set FSC threshold to 0, a negative population showed up, 
whose FSC-SSC both are near to zero.I used 7-AAD as a viability dye, that 
negative population is negative for 7-AAD. Other than that, all the population 
showed a much higher fluorescence intensity compared to negative control.

Here is my direct staining protocal.Could anyone help me figure out what the 
problem might be?
1.Fix w/ 4%PFA 30min on ice,wash;
2.Permiablize w/ 100% methanol drop by drop, incubate on ice 30min; 3.rehydrate with PBS
5min twice; 
4.block w/ 1%BSA+3%Rat/mouse serum(depending on antibody species) 30min RT; 
5.Incubate antibody 1hr RT.
6.Wash 10min twice.

Thank you for your attention!

Yang WANG
doll731@ku.edu
785-864-1842
Department of Molecualr Bioscience
University of Kansas