Following permeabilization, cells were blocked in blocking solution (5% goat serum and 1% BSA in PBS) for 90 min. Samples were then incubated with primary antibodies overnight at 4 C. Primary antibodies were diluted into blocking solution. After 14–16 h of incubation, cells were washed with PBS and then incubated in secondary antibodies. Secondary antibodies were also diluted into blocking solution. Following secondary antibody incubations, samples were washed with PBS. Frequently, samples were treated with a nuclear counterstain [Hoechst 33,342, Invitrogen] for 10 min. Coverslips were mounted in ProLong Gold [Life Technologies].
Images were acquired using a PerkinElmer UltraView Vox spinning disk confocal on a Nikon Eclipse Ti Microscope. Fixed cell experiments were performed on an Apochromat 100x 1.49NA oil-immersion objective, and live-cell experiments were performed on a Plan Aprochromat Lambda 60× 1.40NA oil-immersion objective. Z-stacks were collected at 200nm step-size. Experiments were imaged on either a Hamamatsu EMCCD C9100-50 camera or a Hamamatsu CMOS ORCA-Fusion (C11440-20UP). The EMCCD camera was used with Volocity Software [Quorom Technologies/PerkinElmer]. The CMOS camera was used with VisiView (Visitron).
Images were acquired using a PerkinElmer UltraView Vox spinning disk confocal on a Nikon Eclipse Ti Microscope. Fixed cell experiments were performed on an Apochromat 100x 1.49NA oil-immersion objective, and live-cell experiments were performed on a Plan Aprochromat Lambda 60× 1.40NA oil-immersion objective. Z-stacks were collected at 200nm step-size. Experiments were imaged on either a Hamamatsu EMCCD C9100-50 camera or a Hamamatsu CMOS ORCA-Fusion (C11440-20UP). The EMCCD camera was used with Volocity Software [Quorom Technologies/PerkinElmer]. The CMOS camera was used with VisiView (Visitron).