Immunofluorescence Imaging of HepG2 Cells

Immunofluorescence of HepG2 cells was performed by following the previously described protocol [17 (link)]. Cells were fixed in paraformaldehyde (4% in PBS) and incubated overnight with appropriate antibodies: HMGCR (Abcam, ab242315, dilution 1:100), SREBP-2 (Abcam, ab30682, dilution 1:100), SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-8984, dilution 1:100), SR-B1 (Abcam, ab52629, dilution 1:100), LDLr (Santa Cruz Biotechnology, sc-11824, dilution 1:200), anti-Perilipin-2 (anti-Plin2) antibody (R&D Systems, #MAB76341, dilution 1:100). After incubation with primary antibodies, fixed cells were probed for 1 hour at room temperature with donkey anti-goat secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, Milan, Italy, A-11055), goat anti-rabbit secondary antibody Alexa Fluor 555 (ThermoFisher Scientific, A27039) and goat anti-rabbit secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, A-11008). Coverslips were mounted with Vectashield Antifade mounting medium with DAPI (Vector, H-1200) to visualize nuclear staining. The samples were examined at confocal microscopy (TCS SP8; Leica, Wetzlar, Germany). Images were captured using Leica TCS SP8 equipped with a 40 × 1.40–0.60 NA HCX Plan Apo oil BL objective at RT and Leica LAS X Software.