Figure 4.

Monolayer cultures (upper panel) and aggregates (lower panel) of SAS cells with (F+) or without (F−) filipin III were immunostained for cav1.

For immunofluorescence staining, cultured cells were fixed in 3.5% (w/v) formaldehyde, permeabilized in 0.2% (v/v) Triton x-100, and blocked in 2% (w/v) BSA. The primary antibodies were incubated at 4 °C overnight. Alexa fluor 488-conjugated IgG (Life Technologies) was used as the secondary antibody. After incubation with the antibodies, SlowFade Gold Antifade reagent with 4′,6-diamidino-2-phenylindole (dAPI; Invitrogen/Life Technologies) was added. The specimens were observed using fluorescence microscopy. Nuclei were stained with dAPI.