Af791

Eyes were enucleated and fixed in 4% paraformaldehyde for 2 hours at room temperature. The intact retinas were collected, blocked, and permeabilized in PBS containing 10% goat serum, 3% BSA, 1% Triton X-100, and 0.2% Tween 20 for 1 hour. Samples were then incubated with primary antibodies against rabbit PKM2 (1:100, 4053, Cell Signaling Technology), rabbit Ki67 (1:200, RM-9106, Thermo Scientific), rabbit Fabp5 (1:200, 2.5 μg/mL, RD181060100, BioVendo), rabbit Igf1 (10 μg/mL, AF791, R&D Systems), rat F4/80 (1:100, ab6640, Abcam), rabbit IBa1 (1:400, 019-19741, Sakura Finetek), goat IBa1 (1:400, ab48004, Abcam), rat CD11b (5 μg/mL, 561114, BD Biosciences), and Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–labeled Griffonia simplicifolia isolectin B4 (10 μg/mL; catalogs 121411, 121413, and I32450, respectively; Invitrogen) overnight at 4°C, followed by incubation with fluorescence-conjugated cross-adsorbed secondary antibody (1:500, Molecular Probes, Invitrogen) for 1 hour, and then counterstained with DAPI (Invitrogen). Retinas were flat mounted on microscope slides in a mounting medium (Vectashield; Vector Laboratories) and examined by confocal microscopy (Zeiss 780; Carl Zeiss). For all immunofluorescence experiments, parallel groups of tissues were stained with IgG isotype and secondary antibody as negative controls.

For coculture experiments, OBs were plated onto 6-well plates (20×10³ cells/well). Twenty-four hours later, MKs (20×10³ cells/well) were plated into wells for direct coculture. For indirect coculture, OBs were seeded in the upper chamber of a 0.4 μm transwell insert (Corning, New York, USA) and MKs were seeded in the lower wells. Specified cultures were pretreated with 100 nM of SB431542 (Sigma-Aldrich, St. Louis, Missouri, USA) for OBs for approximately 1 h. MKs conditioned medium (MKs-CM) was obtained by removing MKs via centrifugation (5000 rpm, 10 min). In some assays, neutralizing antibodies against TGF-β1 (9016; R&D, Minneapolis, Minnesota, USA ), VEGF (AF-493; R&D), BMP6 (MAB6325; R&D), IGF-1 (AF791; R&D), PDGF-BB (ab34074; Abcam), CXCl12 (79014; R&D), BMP2 (MAB111; R&D), TGF-β2 (AB-112-NA; R&D), TGF-β3 (20724; R&D), BMP4 (MAB50201; R&D) and IgG (AB-108-C; R&D) were added to the MKs-CM.