Immunofluorescence assays were performed with retinal sections, RPE–choroid flatmounts, or primary RPE cells in 24-well slide chambers. Primary antibodies were used for staining against E-cadherin (3195, Cell Signaling Technology), N-cadherin (13116, Cell Signaling Technology), α-SMA (ab5694, Abcam), RPE65 (GTX13826, GeneTex), METTL3 (ab195352, Abcam), IB4 (DL-1207, Vector Laboratories), F4/80 (MCA497, Abd Serotec), Alexa FluorTM 488 Phalloidin (A12379, Thermo Fisher Scientific), Ki67 (ab15580, Abcam), HMGA2 (8179, Cell Signaling Technology), and GFP (ab290, Abcam).
To evaluate the volume of subretinal fibrosis, RPE–choroid flatmounts were stained with the antibody against Fibronectin (AB2033, Merck Millipore) on Day 28 after laser injury. The volume analysis was performed as previously described (Ye et al., 2015 (link)). Fibronectin was visualized using a confocal laser scanning microscope (TCS SP8; Leica Biosystems). Horizontal optical sections were taken at 1 µm intervals from the top to the bottom of the fibrosis lesion. The area of Fibronectin-positive lesion on each layer was measured using ImageJ software. The total area of each horizontal section was used as an index of Fibronectin volume. All laser spots in each eye were measured.
To evaluate the volume of subretinal fibrosis, RPE–choroid flatmounts were stained with the antibody against Fibronectin (AB2033, Merck Millipore) on Day 28 after laser injury. The volume analysis was performed as previously described (Ye et al., 2015 (link)). Fibronectin was visualized using a confocal laser scanning microscope (TCS SP8; Leica Biosystems). Horizontal optical sections were taken at 1 µm intervals from the top to the bottom of the fibrosis lesion. The area of Fibronectin-positive lesion on each layer was measured using ImageJ software. The total area of each horizontal section was used as an index of Fibronectin volume. All laser spots in each eye were measured.
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