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?
Edited by Eric, 07 July 2010 - 06:22 AM.