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low signal after fixation and permeabilization

siRNA fixation permeabilization colocalization actin

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#1 Mamcy40

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Posted 31 January 2012 - 09:07 PM

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?

Please help.

Mamcy40

#2 bob1

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Posted 06 February 2012 - 01:03 PM

I take it you are keeping the cells in the dark after the transfection to prevent photo-degradation of the 488?





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