antigen masking of actin prior to phalloidin stain

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CATFARTS
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antigen masking of actin prior to phalloidin stain

Hi all,
I have a situation where I am looking at a protein that has a cytoskeletal-like distribution when visualized by IF (looks like it's on stress fibers). When I try applying a phalloidin conjugated fluor after immunostaining for my protein, the actin staining is pretty dim and mostly absent from the regions stained for my protein. Cells not pre-stained for IF have robust staining at the same concentration of phalloidin, and have actin staining in the regions that my protein would presumptively be. I am almost positive that the IF against my protein-of-interest is masking the actin from the phalloidin in the former situation. Does anyone have any creative solutions for establishing colocalization in these circumstances.

Protocol info:
primary Ab: affinity purified Rabbit Ab (1:40) (in house)
Secondary: Gt-a-Rb Alexa 488 (1:200) (Mol Probes)
Phalloidin Alexa 568 (1:500) (Mol Probes)

Cells on cover glass
Fix: 4% PFA in PBS 15 min RT
Permb: 0.1% TX-100, 0.1% BSA in PBS 12 min RT
Block & Ab/phal: 0.1% BSA, 5% NGS in PBS
     block 1 hr RT
     primary O/N @ 4 C  (3x washes)
     secondary 1 hr RT   (3x washes)
     phalloidin 1 hr RT (3x washes)

Any suggestions are greatly appreciated :)

David

Pippuri
Pippuri's picture
 Hi David,

 Hi David,

Have you tried to alter the staining sequence, says first stain with phalloidin, then add Ab against protein of your interest?? I wonder how does it look like...

CATFARTS
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I will be trying that and

I will be trying that and perhaps a couple other variations on the sequence of reagents(e.g. phalloidin and primary together and phalloidin and secondary together) to see what happens.

Thanks for your comment