Apo tirf dic oil immersion objective

Imaging was performed using a Nikon A1 plus confocal microscope with a 60× Apo DIC oil immersion objective with a NA of 1.40 (Nikon Instruments) and appropriate filter sets. Images were acquired with NIS-Elements, version: 4.50.02 (Laboratory Imaging). For TIRF imaging, we used a commercial TIRF system based on a Nikon Ti-E eclipse microscope equipped with a 100× Apo TIRF DIC oil immersion objective with a NA of 1.49 (Nikon Instruments), an iXon Ultra DU-897 EMCCD camera (Andor Technology), and four main laser lines: 405 (Cube, Coherent), 488 (Melles-Griot), 561 (Sapphire, Coherent), and 640 (Cube, Coherent) with corresponding filter sets. Images are usually acquired at a generous TIRF angle to allow imaging of protein stains related to lipid droplets, such as phospho-perilipin-1. Isolated cells were fixed using 4% paraformaldehyde and labeled with antibodies in a buffer containing 1% BSA, 1% goat serum, and 0.05% saponin 1–2 h per labeled antibody. For neutral lipid staining, BODPIY was used in conjunction with confocal imaging. TIRF microscopy was used to detect protein stain only. For imaging of adipocytes, we used previously described protocol (Wasserstrom et al., 2018 ).

HEI-OC1 cells were cultured on glass-bottom dishes (MatTek) and preincubated as described for 2 hours in Krebs-Ringer bicarbonate buffer containing 2 mM glucose, 10 mM HEPES, pH 7.4, 120 mM NaCl, 5 mM NaHCO₃, 5 mM KCl, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 1.2 mM MgSO₄ and 0.2% BSA at 33°C. The cells were stimulated without or with 10 nM insulin for 7 min. Subsequently, the cells were fixed using 4% paraformaldehyde and incubated with antibodies as indicated (1 hour per primary antibody and 1 hour per secondary antibody) in a buffer containing 1% BSA, 1% goat serum and 0.05% saponin. To visualize actin the cells were incubated with phalloidin (Invitrogen) for 1–2 hours.
For total internal reflection fluorescence (TIRF) imaging we used a commercial TIRF system based on a Nikon Ti-E eclipse microscope equipped with a 100×Apo TIRF DIC oil immersion objective with a NA of 1.49 (Nikon Instruments), an iXon Ultra DU-897 EMCCD camera (Andor Technology) and four main laser lines: 405 (Cube, Coherent), 488 (Melles-Griot), 561 (Sapphire, Coherent) and 640 (Cube, Coherent) with corresponding filter sets.