The slides with the tissue were prepared for MALDI imaging by automated matrix deposition (Portrait 630; Labcyte, Sunnyvale, CA). Fifty iterations (one 170-pL droplet per iteration of 10 mg/mL 2′,5′-dihydroxyacetophenone in 70:30 ethanol/water [vol/vol]) were applied across each tissue with a center-to-center droplet distance of 300 μm, and shifts in x or y, or both, were applied so that the final center-to-center spot distance was 150 μm. This procedure ensured no delocalization of analytes to adjacent matrix spots. MALDI data were collected in the positive ion mode at +25 kV accelerating potential on a TOF mass spectrometer (ultrafleXtreme; Bruker) operating in reflector mode with the laser repetition rate set to 500 Hz. Before data collection, a linear external calibration was applied using leucine enkephalin (m/z 556.28; Bruker) and angiotensin II (m/z 1046.54; Bruker). Imaging data sets were acquired over paired (left and right eyes) mouse tissues in the m/z 420 to 1400 range, with a raster step size of 150 μm and 500 laser shots per spectrum. After data acquisition, MALDI images were reconstructed using acquisition and evaluation software (FlexImaging 2.0; Bruker). Each m/z signal was plotted ± 0.5 m/z units. For display purposes, in each individual sample, the data were normalized to total ion current and interpolated (which applies a linear pixel intensity change between adjacent sampling locations for the plotted m/z value), and pixel intensities for all data sets were plotted on the same relative intensity scale (0–80: pink, ∼70; red, ∼50; green, ∼30; light blue, ∼20; dark blue, ∼10) to facilitate signal intensity comparison.