help with cell staining
TomFrey at sprintmail.com
Fri Aug 29 01:51:57 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.
Some IgMs have been published for internal staining, and we have had
some success with suspension staining with one IgM against a nuclear
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
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 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.
More information about the Immuno