help with cell staining

Tom Frey TomFrey at sprintmail.com
Fri Aug 29 01:51:57 EST 1997


Nicole wrote:
> 
> 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.


Some IgMs have been published for internal staining, and we have had
some success with suspension staining with one IgM against a nuclear
antigen.

Not all antigens survive fixation and/or permeabilization.  Formaldehyde
can destroy some epitopes and alcohol fixatives or permeabilizers have a
relatively high incidence of destroying native epitopes.  For
formaldehyde fixed samples, antibodies against native proteins MAY be
more effective, for precipitating/denaturing fixatives antibodies raised
against peptides or known to work in westerns may be best.  (Watch me
grossly over-generalize!)

More information about the antigen and the methods tried would make it
easier for posters to provide to better (more specific) advice.


> 
> >I use biotin horse anti-mouse
> > followed by streptavidin-CY3.  I get background - red specks all over.
> 
> 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

Nicole is giving excellent advice.  Implied in her comments, also, is
that you should have known negative and positive cells type you can
analyze (if at all possible).  I am amazed at how often people just go
for it with a positive target when the antibodies aren't well tested for
the specific application.

Good luck

Tom



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