Sp5 confocal microscope

Eyes were enucleated from P7 and P22 mice, and cornea, lens, optic nerve, and retina tissues were removed. At least 3 animals/eyes per genotype per time point were used. P7 eyes were treated for 5 minutes with PBTx to aid in the separation of the retina from the RPE. RPE was fixed for 45 minutes in phosphate buffered 4% paraformaldehyde. After fixation, RPE samples were rinsed in PBS and incubated in phosphate buffered 3% H₂O₂ for 48 hours at 4°C to quench the RPE autofluorescence. RPE samples were washed in PBS and blocked in 10% Normal Goat Serum (NGS), 1% BSA in PBTx for 2 hours. Primary antibody incubation occurred overnight at 4°C with the antibodies and dilutions to the following antigens: TMEM98 (1:500, 14731-1-AP, Proteintech), MFRP (1:500, af3445, R&D Biosystems, Minneapolis, MN, USA). RPE samples were washed in PBS and incubated with Alexa-conjugated fluorescent secondary antibodies for 2 hours at 22°C. RPE samples were rinsed in PBS and the nuclei were stained with DAPI. A series of 3–4 radial cuts were made to flatten the RPE and underlying scleral and samples were mounted with Prolong Gold Antifade. Images were taken on a Leica SP5 confocal microscope or Olympus BX-51 epifluorescence microscope.

Microscopy was conducted using a Leica SP5 confocal microscope, and images were captured with an Olympus DP27 camera and Olympus cellSens software (version 4.2, Olympus, Tokyo, Japan). The extent of damage to the spheroids was comparatively analyzed.