Cy3 conjugated anti rabbit secondary antibody

Spheroids were fixed in 4% formaldehyde in PBS for 30 min at 4˚C then transferred into 2.0 mL tubes, rinsed three times in 0.1% TBST, permeabilized with 0.5% TBST for 30 minutes on a rotator at RT, rinsed with 0.1% TBST, and incubated in blocking buffer for 1 hour on a rotator at RT. Spheroids were then incubated with mouse anti-β-catenin antibody (1:500, Vanderbilt Protein and Antibody Resource) or rabbit anti-E-cadherin antibody (1:200, Cell Signaling Technology) in blocking buffer overnight on a rotator at 4˚C. After incubation, spheroids were washed three times for 30 minutes per wash in 0.1% TBST and incubated 2–3 hours on a rotator at RT with Cy3-conjugated anti-mouse secondary antibody (1:500, Jackson Immune) or Cy3-conjugated anti-rabbit secondary antibody (1:500, Abcam) diluted in blocking buffer. Spheroids were rinsed in 0.1% TBST then incubated with Alexa Fluor 568 Phalloidin (1:1000; ThermoFisher Scientific) and Hoescht (1:1000) in 0.1% TBST for 20 minutes on a rotator at RT. Spheroids were rinsed 1X in 0.1% TBST. Spheroids were put into 1X PBS (Vanderbilt Molecular Biology Resource) then mounted on microscope slides with ProLong Gold Antifade Reagent (Invitrogen). Images were acquired using a Nikon Spinning Disk microscope with Andor DU-897 EMCCD camera with 561 nm and 405 nm lasers. Images were processed using ImageJ.