Slit lamp

Induction of laser-induced choroidal neovascularization (LCNV) was performed in six adult C57BL/6 mice, three of each sex, following published protocols (20 (link), 21 , 34 (link)). Briefly, following anesthetization and pupillary dilation, four laser-induced choroidal lesions were created in each eye by rupturing the Bruch’s membrane with a solid-state laser photocoagulator, GYC-500 (Green photocoagulation 532 nm laser) mounted on a slit-lamp (Nidek, Fremont, CA, USA). Lesions were created using the following laser parameters: 100 μm spot size, 0.1 s duration, and 0.1 Watts. Rupture of the Bruch’s membrane was confirmed using OCT imaging (Phoenix Research Laboratories, Pleasanton, CA, USA) of the mouse LCNV as shown in Supplementary Figure 6. Four days later, the LCNV mice were injected intraperitoneally with 10 mg/kg InflammaProbe-1 or an equimolar dose of Oregon Green 488 cadaverine, 5-isomer (5.46 mg/kg) in 100 μL PBS with 10% DMSO. Brightfield and fluorescent fundus images were acquired 6 h post-injection using the Micron IV retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA, USA). Annotations were added to both images using PowerPoint (Microsoft, Redmond, WA, USA). The images were representative of 12 eyes.

To induce CNV in C57BL/6 mice, laser-induced ruptures of the Burch's membrane were performed with an 532 nm green laser photocoagulator mounted on a slit-lamp (Nidek Inc.; San Jose, CA). Four lesions were created in both the left and right eyes of each mouse. Laser parameters used were 100-μm spot size, 0.1-s duration, and 0.3 Watts. On day-3 post-laser treatment, mice were divided into two groups and received systemic injections of AS-Eng shRNA–lipid at a concentration of 0.5 mg/kg in PBS. Eighteen hours after the probe-injection, the mice were sacrificed and the eyes were fixed in neutral buffer formalin (NBF) for 30 min. The anterior segments and lenses were removed while submerging the eye in NBF solution. The choroid-Bruch’s membrane-RPE complex was dissected as previously described^{40 (link),41 (link)} and tissues were stained and analyzed for IBA1 and IB4, as described above.