Elements general analysis

Eyes were carefully enucleated and placed in ice-cold PBS. Eyes were dissected in EGM2-MV supplemented (hydrocortisone was omitted) medium (CC3202; Lonza, Walkersville, MD, USA) into posterior eye cups containing sclera, RPE, and choroid complex. The peripheral choroid was separated from the central choroid, cut into 0.5 mm × 0.5 mm pieces, and placed into growth factor reduced Matrigel (#356231; Corning, Bedford, MA, USA) in a 48-well plate on ice. The Matrigel was solidified by incubating at 37°C for 10 minutes. EGM2-MV medium was changed every 2 days. Sunitinib (PZ0012) and propranolol (P0884) were purchased from Sigma (St. Louis, MO, USA). Sunitinib was added on Day 0, and propranolol was added on either Day 0 or Day 2. Pictures were taken on a Nikon Ti2 Widefield microscope (Buffalo Grove, IL, USA) using a 4× objective and Nikon NIS Elements software. Images were analyzed with the Nikon Elements General Analysis. Images were preprocessed with edge detection and segmented with thresholding. The largest area was measured for each image. The central choroidal tissue area was subtracted from the total angiogenesis area.

Parietal cells and chief cells were quantitated as previously described¹⁷ (link) using confocal micrographs captured using a 20× microscope objective and 1-μm–thick optical sections. Cells were counted using the ImageJ (National Institutes of Health, Bethesda, MD) count tool. Cells that stained positive with anti-H+/K+ antibodies were identified as parietal cells, while cells that stained positive with anti-MIST1 antibodies and were GSII negative were identified as mature chief cells. Counts were reported as the number of cells observed per 20× field. Images were chosen that contained gastric glands cut longitudinally. Leukocytes were quantitated using Nikon Elements General Analysis (Nikon, Tokyo, Japan). Six tile-scanned images were captured using the 20× objective and stitched on Zen Black. Eosinophils were identified as CD45/Siglec F double-positive, while macrophages were CD45/CD68 double-positive.