Lsm 5 live duo highspeed spectral confocal system

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.

Following C1-Bodipy 500/510 C12 labeling, the RPE was permeabilized with 0.1% SDS, blocked by incubation for 20 min with 10% fetal calf serum, and incubated overnight with a 1:500 dilution of rabbit anti-ZO-1 antibody (Invitrogen 40–2200). The RPE was washed and incubated for 4 h at room temperature (RT) with Alexa-647-conjugated anti-rabbit secondary antibody diluted in blocking buffer. The RPE was gently rinsed in PBS and labeled for 5 min with a 1:1000 dilution of 4′, 6-diamidino-2-phenylindole (DAPI), then rinsed 5 times in PBS for 5 min at RT and mounted in DAKO mounting medium. Confocal imaging of Bodipy C12/ZO-1 fluorescence was performed with a Zeiss LSM 5 LIVE DUO High-speed/Spectral Confocal system. Images were acquired using Zeiss Zen software.

Mouse NR primary cells or ARPE‐19 cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature (RT), permeabilized in 0.1% triton for 10 minutes at RT, saturated 20 minutes in 0.1% SDS + 10% donkey serum in PBS for 20 minutes at RT and incubated overnight at 4°C with either mouse monoclonal anti‐Rhodopsin antibody RET‐P1 (Novus Biologicals^®, NBP120‐3267 diluted to 1:500) or rabbit monoclonal anti‐Nrf2 antibody (clone D1Z9C, ref: 12721; Cell Signaling TECHNOLOGY^® diluted at 1:200), and detected with Alexa594‐conjugated anti‐rabbit or Alexa488‐conjugated anti‐rabbit secondary antibodies, respectively. Confocal imaging of Rhodopsin and Nrf2 translocation to the nucleus was performed with a Zeiss LSM 5 LIVE DUO Highspeed/ Spectral Confocal system. Image acquisitions were obtained with the Zeiss Zen software.