Aqueous angiography was performed as described before.5 (link) Eyes were trimmed of extraocular tissue, oriented by inferior oblique insertion location,19 and pinned to Styrofoam. A Lewicky Anterior Chamber (AC) Maintainer (BVI Visitec, Alcester, UK) was inserted through a 1-mm side port (Alcon, Fort Worth, TX, USA) into the anterior chamber. Balanced salt solution (BSS; Alcon) was introduced for a 1-hour preperfusion period at room temperature (RT) with a reservoir height set at 5 inches above the eye to provide a gravity-delivered pressure of ∼10 mm Hg as previously described. Eyes were kept moist with RT BSS-soaked gauze. Simultaneously, 25% fluorescein (Akorn, Lake Forest, IL, USA) was diluted at RT in BSS to 2.5%. Indocyanine green (ICG; I2633; Sigma-Aldrich Corp., St. Louis, MO, USA) was dissolved with water into a 2% stock solution, and ICG was subsequently diluted in BSS to 0.4%. These concentrations were chosen because they have been described for clinical use in live humans as intraocular capsular stains for cataract surgery.20 (link)In most cases, ICG (n = 11) was first introduced for aqueous angiography at 10 mm Hg followed by fluorescein aqueous angiography in the same eye as previously done in cows (Saraswathy S, et al. IOVS 2015;56:ARVO E-Abstract 247). Alternatively, 3-kD fixable and fluorescent dextrans (Life Technologies, Carlsbad, CA, USA; diluted to 2.5 mg/mL in BSS) were used (n = 2) at 10 mm Hg.5 (link),21 (link),22 (link) The eyes were placed in front of the Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany; fluorescein capture mode: excitation wavelength = 486 nm and transmission filter set at >500 nm; ICG capture mode: excitation wavelength = 786 nm and transmission filter set at >800 nm) with fluorescent images taken with a 55° lens using a 25-diopter focus. Confocal scanning laser ophthalmoscopic (cSLO) infrared images were taken to center the eye. Prior to tracer application, cSLO fluorescent angiographic images using the fluorescein or ICG capture mode were taken to provide a standard pretracer intensity background image, which appeared black. Subsequent fluorescein or ICG capture mode images were taken at various time points in various positions or face-on after tracer introduction. To prevent image signal intensity saturation over time during prolonged imaging sessions, the laser sensitivity setting on the Spectralis was adjusted with each image to set the central fluorescent signal in the anterior chamber to just under signal saturation.