Mab5428

Immunohistochemistry was performed on rhesus macaque eyes as described previously.¹³ (link) Briefly, eyes were fixed with 4% paraformaldehyde (PFA) on necropsy, and anterior segments, lens, and vitreous were dissected out within 30 minutes of collection. The remaining posterior eye cups were fixed with 4% PFA for 2 hours at room temperature and washed with phosphate buffered saline (PBS) 4 times for 15 minutes. Followed by cryoprotection with 30% sucrose overnight at 4°C, tissues were embedded in optimal cutting temperature compound and cryosectioned at 18-µm thickness. Consecutive sections enabled comparison of native AF with fluorescence immunolabeling using anti-C5b9 antibody (ab55811; Abcam, Cambridge, MA, USA) to label druse contents, and anti-RPE65 antibody (mab5428; Millipore, Burlington, MA, USA) to label RPE cells. For AF detection, the section was mounted with mounting media after washing with PBS 3 times for 5 minutes. For immunohistochemistry, sections were blocked with 10% normal donkey serum for 30 minutes, then incubated in primary antibody for 1 to 2 hours at room temperature, followed by detection with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured using a confocal microscope (FV1000; Olympus America, San Jose, CA, USA).

Eyes were enucleated and placed in 4% paraformaldehyde (PFA) for less than 30 min, followed by gentle removal of anterior segments, including lens and vitreous. Whole eye cups were fixed in 4% PFA for 2 h at room temperature, rinsed with phosphate-buffered saline (PBS), cryoprotected with 30% sucrose overnight, embedded in optimal cutting temperature (OCT) medium, and then cryosectioned (18 μm) using a cryostat (CM3050 S, Leica). For immunohistochemistry, sections were washed with PBS, blocked with 10% normal donkey serum for 30 min, then incubated in primary antibody for 1–2 h at room temperature, followed by Alexa Fluor 568-conjugated secondary antibodies (Invitrogen). Primary antibodies include RPE65 (MAB5428, 1:250, Millipore), EGFP (Novousbio, NB100-1770, 1:500), IBA-1 (Wako, AB10558, 1:100), GFAP (Dako, Z0334, 1:200), CD45 (BD, 552566, 2.5 μg/mL), and CD3 (Abcam, ab11089, 1:100).