Sample preparation for confocal and TIRF microscopy

HeLa cells cultured on fibronectin-coated 13-mm coverslips were washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. For detection of intracellular proteins, cells were also perme–abilized with 0.2% Triton X-100 for 10 min before antibody incubation. Cells were incubated with primary antibodies for 2 hours, appropriate secondary antibodies were conjugated to Alexa Fluor 568 or Alexa Fluor 647, and phalloidin was conjugated to Alexa Fluor 568 or 647 for 1 hour. Cells were mounted onto slides using Immunofluore (ICN).

For TIRF analysis, HeLa cells cultured in fibronectin coated eight-well glass bottom chambers (Ibidi) were washed with PBS and fixed with 4% PFA in PBS for 10 min. For detection of intracellular proteins, cells were also permeabilized with 0.2% Triton X-100 for 10 min before antibody incubation. Cells were incubated with primary antibodies for 2 hours, and appropriate secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 568. For TNF546 labeling, TNFR1-GFP–transfected SH3 were incubated on ice for 20 min with TNF-546 (10 ng/ml) in PBS before fixation with 4% PFA. TIRF images were acquired in PBS using a Nikon A1R microscope with TIRF capability using a CFI Apo TIRF with 1.48 numerical aperture (NA) 60× oil objective (Nikon) and a cooled charge-coupled device (CCD) camera (Hamamatsu).