*2.3. Grading of Immunostaining*

Three independent masked observers, using a previously described grading system [**? ?** ], graded the intensity of the immunoreactivity for each antibody in transverse retinal sections. The intensity of labeling was graded qualitatively as: 10, strong bright intense immunoreactivity, 9, bright intense; 8, uniformly intense; 7, patchy and intense; 6, uniform and moderate; 5, patchy and moderate; 4, uniform and weak; 3, patchy and weak; 2, uniform and very weak; 1, patchy and very weak; and 0, none. A mean score ±SEM for each group was determined from the scores of all graders for each retina structure.

### *2.4. Trypsin Digestion and the Detection of Superoxide*

The retinal vasculature was prepared as previously described [**?** ]. Briefly, mouse eyes were fixed overnight in 4% paraformaldehyde, freshly made in PBS. The retinas were dissected from the eye cups, kept in water overnight, and digested in 3% trypsin (Invitrogen-Gibco, Grand Island, NY, USA) for 3 h at 37 ◦Celcius. The tissue was mounted carefully on a glass slide under a dissection microscope. The tissue was stained with PAS-H&E (periodic acid Schi ff–hematoxylin and eosin; Gill No.3; Sigma-Aldrich), according to the instruction manual. The images were taken using a Zeiss light microscope equipped with a digital camera (AxioCam MRC5, Axiovert 200; Carl Zeiss Meditec, Inc., Dublin, CA, USA), using 20X objective lenses. Eight to ten representative fields from each quadrant of the retina were imaged and the number of acellular capillaries per square millimeter of retina were quantified.

For detecting the reactive oxygen species (ROS), the superoxide indicator, hydroethidine, was used to detect the production of superoxide radicals, as previously described [**?** ]. Superoxide oxidizes hydroethidine to yield a red fluorescent signal at approximately 600 nm. Mice received two intraperitoneal injections, 15 min apart, of freshly prepared hydroethidine (20 mg/kg; Invitrogen) and were euthanized 18 h after injection. The fluorescence intensity was measured in the neural retina using a fluorescent plate reader (BioTek, Winooski, VT, USA) and a spectrofluorometer (FLUOstar Optima; BMG Labtechnologies, Cary, NC, USA). The relative fluorescence intensity was calculated by normalizing to protein concentration.
