Immunofluorescence

Micropatterned coverslips were fixed with 4% paraformaldehyde (Electron Microscopy Sciences 15713) in warm medium for 30 min, rinsed three times with PBS−/−, and blocked and permeabilized for 30 min with 3% normal donkey serum (Jackson ImmunoResearch 017-000-121) and 0.5% Triton X-100 (Sigma-Aldrich 93443) in PBS−/−. Specimens were incubated with primary antibodies for 1.5 hr at room temperature, washed three times for 5 min each in PBS−/−, incubated with secondary antibodies conjugated with Alexa 488, Alexa 555, Alexa 594, or Alexa 647 (Molecular Probes) at 1/1000 dilution for 1 hr at room temperature. After 30 min incubation with 100 ng/ml 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific D1306), specimens were washed three times with PBS−/−. Coverslips were mounted on slides using ProLong Gold antifade mounting medium (Molecular Probes P36934).