Retinal Vascular Pericyte Analysis

The eyes were rapidly enucleated and fixed in 4% paraformaldehyde (PFA) for 2 min before being dissected in flower shape in 2× phosphate-buffered saline (PBS). The retinae were fixed again in 100% methanol and stained with primary antibodies against CD31 (rat monoclonal, 553370, BD Biosciences, San Jose, CA, USA, RRID:AB_394816) and NG2 (rabbit polyclonal, AB5320, Merck Millipore, Germany, RRID:AB_11213678) overnight at 4 °C, followed by incubation with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (A-11006 or A-11012, Thermo Fisher Scientific, USA) and 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, USA). The retinae were flatmounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, USA) and examined by confocal microscopy (Zeiss LSM 800, Zeiss, Germany). The retinal vasculature was analyzed using AngioTool [62 (link)]. Pericyte coverage was determined by the ratio of NG2⁺ area/CD31⁺ area.