Alexa fluor 594 and 488 conjugated secondary antibodies

Immunofluorescence assays were performed on choroidal flat mounts. Briefly, after being fixed, the samples were blocked with 0.3% Triton X-100 and 5% goat serum albumin (Beyotime, Shanghai, China) in PBS for 1 h at room temperature. The tissues were immunostained with primary antibodies against F4/80 (1:500; CST, Beverly, MA, USA), Ym-1 (1:500; Stem Cell Technology, Vancouver, Canada) and isolectin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C, washed three times with PBS, and then stained for 45 min at 37 °C with Alexa Fluor 594- and 488-conjugated secondary antibodies (1:1000; Proteintech, Chicago, IL, USA). The nuclei were stained with 4,6-diamidino-2-phenylindole. Images were visualized using a fluorescence microscope (Olympus, Center Valley, PA, USA) and a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany) [20 (link)].

Immunocytochemistry assays were performed on retinal sections or in 24-well slide chambers. Briefly, after fixation, the samples were blocked with 0.3% Triton X-100 and 5% goat serum albumin (Beyotime) in PBS for 1 h at room temperature. Tissue sections were immunostained with primary antibodies against RelA (1:300, Cell Signaling Technology), RelB (1:300, Abcam), c-Rel (1:300, Abcam) and RPE65 (1:300, Novus Biologicals, Littleton, CO, USA) overnight at 4 °C. The samples were washed with PBS three times and then were stained for 45 min at 37 °C with Alexa Fluor 594- and 488-conjugated secondary antibodies (1:1 000, Proteintech). The nuclei were marked by 4′,6-diamidino-2-phenylindole. RPE cells were visualized using a fluorescence microscope (Olympus) and a Leica TCS SP8 confocal laser scanning microscope (Leica TCS NT, Wetzlar, Germany).
For ultrastructural analysis, the samples were send to Shanghai FuDan University School of Medicine after fixation in 2.5% glutaraldehyde (dissolved in PBS) at 4 °C. The ultrathin sections were observed under an electron microscope (Tecnai G2 spirit twin, FEI, Eindhoven, The Netherlands).