Following euthanasia of the mice, eye balls were enucleated immediately and dissected. The eyecups were fixed in 4% paraformaldehyde for 7 h, and then cryoprotected in 30% sucrose in PBS for another 14 h, all at 4 °C. Cryosections of 10 μm each were cut from the eyecups frozen in the optimum cutting temperature (O.C.T.) embedding medium (Sakura Finetek 4583, Sakura Finetek USA, Inc., Torrance, CA), and used for immunostaining following our method described previously42 (link) with minor modifications. Briefly, retinal sections were permeabilized with 1% Triton X-100 in PBS for 20 min, blocked with 10% normal goat serum (Cat#71–00–27; Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 2 h at room temperature, and then incubated with purified rabbit anti-sigma-1 receptor antibody31 (link) (1/150 dilution) and mouse monoclonal anti-synaptophysin (Cat. #MAB368, 1/500 dilution; Chemicon, Los Angeles, CA) overnight at 4 °C. After rinsing the sections 3×, secondary antibodies (Alexa-488 conjugated goat-anti-rabbit and Alexa-594-conjugated goat-anti-mouse) at 2 μg/ml was applied at room temperature for 2 h. Sections were then rinsed 3×, counterstained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min, and then mounted in the Prolong Gold mounting medium (Invitrogen, Carlsbad, CA) and cover-slipped. The slides were left in the dark overnight and then sealed using clear nail polish (Electron Microscopy Sciences, Hatfield, PA). Images were then taken with a Nikon A1R laser confocal microscope (Nikon, Tokyo, Japan) supplied with a green 488 nm Argon laser and a red 561 nm DPSS laser through an Apo60X VC oil-immersion objective with NIS elements software23 (link). Z-stacks were collected at 0.5 μm each, for a total thickness of 10 μm. Sequentially collected images were stacked together using the ImageJ program.
The porcine, bovine, monkey, and human retina sections were prepared similarly. The porcine and bovine eyeballs were obtained from the Black Earth Meats slaughter house in Cross Plain, WI, USA. The monkey retinal sections were from the Primate Research Center, University of Wisconsin, Madison, WI, USA. The human eyeball was procured from the Minnesota Lion Eye Bank.
The porcine, bovine, monkey, and human retina sections were prepared similarly. The porcine and bovine eyeballs were obtained from the Black Earth Meats slaughter house in Cross Plain, WI, USA. The monkey retinal sections were from the Primate Research Center, University of Wisconsin, Madison, WI, USA. The human eyeball was procured from the Minnesota Lion Eye Bank.
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