Visualization of Caveolae and Endocytic Regulators

Induced Dyn2-GFP-Cav1-mCh HeLa cells were transfected with the EHD2-BFP expression vector and seeded on precision coverslips (No. 1.5H, Paul Marienfeld GmbH and Co. KG) in 24-well plates at 50 × 10³ cells/well and incubated overnight (37°C, 5% CO₂). Following incubation with Dyngo 4a (30 μM) or DMSO (0.001%) for 30 min, cells were fixed with 3% PFA in PBS (Electron Microscopy Sciences) and subsequent permeabilization and blocking was carried out simultaneously using PBS containing 5% goat serum and 0.05% saponin. Cells were then immunostained with rabbit anti-PTRF, RRID:AB_88224 (Abcam) followed by goat anti-rabbit IgG secondary antibody coupled to Alexa Fluor 647, RRID:AB_2535814 (Thermo Fisher Scientific) as previously described (Lundmark et al., 2008 (link)). Confocal images were acquired using the Zeiss Spinning Disk Confocal microscope (63× lens). Micrographs were prepared using Fiji (Schindelin et al., 2012 (link)) and Adobe Photoshop CS6.