Mab3494

Nile Red staining of lipids [54 (link)] was used alone or in conjunction with fluorescent protein immunostaining. The eyes of 3-month-old mice were enucleated and the retina was separated from the RPC/choroid layer to obtain an empty eyeball [31 (link)]. The eyes were fixed in 4% PFA for 1 h at RT, washed with PBS, and permeabilized with 0.1% sodium dodecyl sulfate. They were then incubated for 20 min at RT in blocking buffer (10% fetal calf serum in PBS), and incubated overnight at 4°C with a mouse anti-mouse ATP synthase (Millipore MAB3494, 1:500) or rabbit anti-mouse perilipin (D1D8, Cell Signaling Technology # 9349, 1: 200) primary antibody. The tissues were then incubated for 4 h at RT with Alexa Fluor 488-conjugated anti-rabbit or anti-mouse secondary antibodies diluted in blocking buffer. The immunostained eyeballs were gently rinsed in PBS, incubated with Nile Red solution (10 μg/ml) for 30 min at RT in the dark, washed twice in PBS for 5 min at RT, and incubated with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000) for 5 min at RT. The eyeballs were finally rinsed 5 times in PBS for 5 min at RT and mounted in Dako mounting medium. Confocal imaging was performed with a Zeiss LSM 5 LIVE DUO Highspeed/Spectral Confocal system. Images were acquired with Zeiss Zen software, and LDs were counted with ImageJ software.