Immunofluorescence Analysis of Ocular Tissues

Immunofluorescence analyses were performed as described previously^{15 (link),47 (link)}. Paraffin sections of fibrovascular tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10-mM citrate buffer (pH 6). Mouse eyeballs were fixed in 4% paraformaldehyde for 30 minutes on ice, incubated in PBS solution with increasing concentration of sucrose (10, 20, and 30%), and embedded in Frozen Section Compound (Leica, Exton, PA). Sections were probed with primary antibodies for galectin-1, AGE, CD45, CD68, GFAP, GS, Iba-1, IL-1β, IL1R1 and TLR4 followed by secondary antibodies. Nuclei were counterstained with DAPI, and sections were visualized under a Biorevo microscope (Keyence, Tokyo, Japan).