Trouble staining for Fluorescent Microscopy - unspecific labeling (Feb/14/2007 )

Hello !

I am detecting a protein in primary cells (from mice) by immunoflourescence microscopy.

The problem is that these mice are knocked out for that protein, so I shouldn't see the staining in the fluorescent microscopy!
Does anyone know why is this happening to me?

I have already reduced the primary Ab concentration and changed the blocking solution (to goat serum).

I use the same antibody to detect the protein by western-blot in wild type cells and it works perfectly.

I would be very grateful if anyone could post the protocols that they use for immunofluorescence detection!!!!
I think there could be a problem with my technique (cell fixation, permeabilization, ...)

Thanks very much.

Hello !

I am detecting a protein in primary cells (from mice) by immunoflourescence microscopy.

The problem is that these mice are knocked out for that protein, so I shouldn't see the staining in the fluorescent microscopy!
Does anyone know why is this happening to me?

I have already reduced the primary Ab concentration and changed the blocking solution (to goat serum).

I use the same antibody to detect the protein by western-blot in wild type cells and it works perfectly.

I would be very grateful if anyone could post the protocols that they use for immunofluorescence detection!!!!
I think there could be a problem with my technique (cell fixation, permeabilization, ...)

Thanks very much.

also, like westerns, you need to block and wash, etc, properly...also might want to tone down your secondary/detection

perhaps it would be easier if you posted YOUR protocol, and we can see if we can find errors?