Phalloidin 488 a12379

The protocol for fluorescence microscopy was followed as previously published [18 (link)]. Additionally, the primary antibodies used were: HA (cst-3724; Cell Signaling), β-catenin (sc-7963; Santa Cruz); while the secondary antibodies: Alexa flour 568 #A11036, and phalloidin 488 #A12379 (Thermo Scientific).

We followed previously published protocol by our laboratory for fluorescence microscopy [31 (link)]. The DVL2 primary antibody was used at a 1:200 dilution (LSBio#375617); while the secondary antibodies: Alexa flour 568 #A11036 and phalloidin 488 #A12379 were used at 1:300 dilution (Thermo Scientific). Images were taken by Nikon confocal TIE inverted microscope at a 60X magnification.

3.5 × 10⁵ cells were seeded onto coverslips (12 mm) in a 60 mm tissue culture dish. The cells were fixed with 4% paraformaldehyde for 15 mins/RT, followed by quenching with 50 mM ammonium chloride (NH₄Cl), permeabilization with 1% Triton X-100 for 10 mins. The coverslips were blocked with 5%BSA for 30 mins, followed by 1 hr incubation with the primary antibody, HA (cst-3724; Cell Signalling). Samples were then incubated with secondary antibodies: Alexa flour 568 #A11036, and phalloidin 488 #A12379 (Thermo Scientific) for 1 hr/RT. The samples were rinsed in PBS for 5 mins and mounted with prolong gold antifade mounting solution with DAPI (P36941, Thermo Scientific), then cured overnight at RT and stored at −20 °C until imaged. The samples were imaged using a laser scanning confocal microscope Nikon T-1E with a 60x objective and NIS software.