help with cell staining

Nicole nglowack at students.wisc.edu
Thu Aug 28 12:08:18 EST 1997


Gabriel ,
> I have been trying to stain fixed/permeabilized cells grown on coverslips
> with IgM mouse monoclonal antibodies.  

Are you trying to stain for intra- or extra-cellular antigens?  IgM is a
bad choice for intra-cellular staining.  It's just too big to
effectively get into a permeablized cell.  

I use biotinylated horse anti-mouse
> followed by streptavidin-CY3.  I have tried a number of dilutions of all
> the reagents involved but I get background - red specks all over the cells.

Can you do this in fewer steps?  Each step has the potential for its own
background staining.  What do your controls look like?  Isotype
control?  Biotin HAM, and possibly even the strep-av CY3 will
nonspecifically bind to the cell.  

Stain with your usual protocol, just the biotin HAM followed by strep-av
-CY3 and just the strep-av-CY3 to check background.  

What kind of cells are you staining?  This may affect the "stickiness"
of the antibodies.

Do you see increased staing in a known positive?  If so then this may
just not be a very sensitive system.  If not, then the problem may be
more serious.

Nicole



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