Diffused protein in IF stainning - (Jul/07/2010 )

Hi guys,

I encountered a weird problem in IF staining for actin filament using TRITC-phalloidin. I fixed cells in 4% paraformaldehyde for 10min and then permeabilize cells with cold methanol for 5 min. Then I just follow the standard procedure proceeding to the secondary antibody incubation. Then I use 1ug/ml TRITC-phalloidin, incubation time is less than 30min. I couldn't find anything wrong in the protocol but then I can't get any filament staining but rather diffused staining. Could anyone know any clue why this happened?

I have a feeling they are stained well. They just look very packed. could this be because of your microscope? Also, better use Triton X100 for permeabilization. don't use methanol alone.

check Abcam's protocol for IF

Curtis on Jul 8 2010, 09:22 AM said:

Thanks for the suggestion. The reason why I use methanol is I also stained for a membrane receptor. I am afraid Triton X100 will probably disrupt membrane protein, so I chose for methanol. And for the actin staining, I think it's quit ugly. I didn't see any fiber at all. And if there is no fiber, it shouldn't be stained, right?