Pericyte Labeling after Laser Injury

Seven days after laser injury, eyes were enucleated and fixed in 4% para-formaldehyde for 1 h on ice, incubated in an increasing concentration of PBS/sucrose (5, 10, and 20%), and embedded in frozen section compound (Leica, Exton, PA, USA). After blocking, the sections were incubated with rat anti-PDGFR-β (1:100, Thermo Fisher Scientific) and rabbit anti-NG2 antibody (1:100, Millipore, USA) and subsequently Alexa Fluor 546 secondary antibodies (1:200, Molecular Probes, Waltham, MA, USA). The specimens were mounted in mounting medium, including DAPI (1:500, Vector Laboratories, Burlingame, CA, USA), and examined under a fluorescence microscope (BIOREVO, Keyence, Tokyo, Japan). The number of nucleus surrounded by NG-2 positive signals, corresponding to pericytes, are counted unbiasedly in five consecutive tissue sections, as previously described [17 (link),18 (link)].