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

Chin Fen Teo
Chin Fen Teo'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