Alexa flour 568 a11036

8 × 10⁵ cells were seeded onto coverslips (12mm) in a 60mm tissue culture dish. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by a wash with PBS for 5 minutes, a quench step with 50mM ammonium chloride (NH₄Cl) in PBS for 5 minutes with an additional 5 minutes PBS wash. The coverslips were blocked with 5% Bovine serum albumin (BSA) in PBS (blocking buffer) for 30 minutes, followed by an hour incubation with the following primary antibodies in 5% BSA in PBS: DVL-1 (D3570; Sigma) and DVL-3 (SAB4200007; Sigma). The samples were rinsed 3 times with PBS and then incubated with secondary antibodies purchased from ThermoFisher scientific (Alexa flour 568 #A11036, Alexa fluor 647 #A21235 and Alexa fluor phalloidin 488 #A12379 from Thermo Scientific) for 1 hour at room temperature. The samples were rinsed several times in PBS for 5 minutes each and then mounted with prolong gold antifade mounting solution with DAPI (P36941, Thermo Scientific), then cured overnight at room temperature 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.

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.