low signal after fixation and permeabilization - (Jan/31/2012 )
Please advice me on this.
I am transfecting my cells with alexa488 siRNA lipoplexes. Once I fix the cells (I tried 2.5 to 4% paraformaldehyde with 15-20 min incubation), the signal reduces with time. After actin phalloidin cell staining, that requires permeabilizing the cells, I see no green signal in the microscope (epi and confocal).
Actually I want to do colocalization studies of taken up siRNA with actin. How do I make this work? I know I cant use phalloidin for live cells. IS there any other way to stain actin in live cells except for using the method of transient transfection for actin?
I take it you are keeping the cells in the dark after the transfection to prevent photo-degradation of the 488?