The unroofed cells were incubated in 3% bovine serum albumin/PBS (BSA, m/v, fresh, Fisher Bioreagents #BP9703) for 1.5 h, followed by primary antibody (1:100 in 3%BSA/PBS) incubation for 1 h. Next, cells were washed thoroughly with PBS and the secondary antibody tagged with fluorescence dye (1:500) or GFP-nanobody (1:500) was applied for 1 h. Afterwards, cells were washed 4 times in PBS and stored in fresh PBS at 4 C until the samples were imaged. The following antibodies were used: anti-Caveolin1-Rabbit (abcam #ab2910), anti-Caveolin1-mouse (Santa Cruz #sc-53564), anti-Cavin1-Rabbit (abcam #76919), anti-Cavin2-Rabbit (abcam #ab76867), anti-Cavin3-Rabbit (abcam #abcam2912), anti-EHD2-goat (abcam #ab23935), anti-Pacsin2-Rabbit (Proteintech #10518-2-AP), anti-EHBP1-Rabbit (Proteintech #17637-1-AP), anti-mouse-Clathrin heavy chain (Thermo-Fisher #MA1-065, 1:2000), anti-mouse-Dynamin2 (Santa Cruz, C-18; #sc-6400), anti-rabbit-Atto647N (Rockland #611-156-122), anti-goat-Atto647N (Rockland, #610-156-121), anti-goat-Atto647N (Rockland #605-456-013 S), anti-rabbit-Alexa568 (Invitrogen #A11036), Fab2-anti-rabbit-Alexa594 (ThermoFisher #A-11072), Fab2-anti-mouse-Alexa488 (ThermoFisher #A-11017), Fab2-anti-mouse-Alexa568 (ThermoFisher #A-11019), GFP-nanobody-Atto647N (Chromotek #gba647n-100), Phalloidin-Alexa488 (ThermoFisher #A12379).
Matthaeus C., Sochacki K.A., Dickey A.M., Puchkov D., Haucke V., Lehmann M, & Taraska J.W. (2022). The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane. Nature Communications, 13, 7234.
+ Open protocol