TIRF microscopy images were acquired on an inverted Nikon Eclipse Ti-E microscope, equipped with a 100 × 1.49 NA objective (Nikon), an iXon EMCCD camera (Andor), laser lines at 405, 488, 561, and 647 nm (Agilent), a multiband pass ZET405/488/561/647× excitation filter (Chroma), a quad-band ZT405/488/561/647 dichroic mirror (Chroma), and appropriate emission filters for imaging of mRFP (600/50 nm, Chroma) and GFP (525/50 nm, Chroma). Illumination was performed by TIRF to ensure exclusive illumination of the plasma membrane.
For ICS, the cells are fixed with 100% methanol at −20°C for 10 min. After washing with PBS, they were permeabilized with 1% Triton X-100 at 37°C for 30 min. After blocking, add primary C-terminus anti-PANX1 (Santa Cruz Biotech.) or N-terminus anti-PANX1 (Alomone Labs) was added and incubated at 4°C at 12 h followed by addition of second anti-mouse-PE (Santa Cruz) at room temperature for 30 min. After washing, the cover slips containing cells were mounted and observed using a confocal microscope (Leica).
For ICS, the cells are fixed with 100% methanol at −20°C for 10 min. After washing with PBS, they were permeabilized with 1% Triton X-100 at 37°C for 30 min. After blocking, add primary C-terminus anti-PANX1 (Santa Cruz Biotech.) or N-terminus anti-PANX1 (Alomone Labs) was added and incubated at 4°C at 12 h followed by addition of second anti-mouse-PE (Santa Cruz) at room temperature for 30 min. After washing, the cover slips containing cells were mounted and observed using a confocal microscope (Leica).