Phosphor Imaging Autoradiography

For digital Phosphor Imaging Autoradiography (PI-ARG) of atherosclerosis, frozen 8 μm sections of mouse aortic roots were mounted on glass slides, and aortas from both Apoe^–/– and WT mice were pinned up on a thin foam pad and placed on an imaging plate (BAS-IP-2025 and −2040 SR storage phosphor screens, GE Healthcare Life Sciences, Pittsburgh, PA, USA). After an exposure period (3–5 days, depending on the experimental setup), the imaging plates were read at a pixel resolution of 50 μm with an Amersham Typhoon FLA 9500 Phosphor Imager (GE Healthcare Life Sciences). ImageQuant TL 8.2 software (GE Healthcare Life Sciences) was used for quantitative analysis of ⁸⁹Zr-tracer uptake in the aorta. ROIs were manually drawn on each aorta, including whole aorta (WA), aortic arch, thoracic aorta (TA), and abdominal aorta (AA). The plaque ROIs were outlined in the aortic arch, guided by a corresponding image of full-length aorta stained with Sudan IV. Negative control ROIs were determined for both thoracic and abdominal parts of aortas as a nonplaque area within the aorta with the lowest activity. The background counts for the ROIs were outlined on each imaging plate. The threshold intensity values were set by estimating the lowest (lightest) pixel within the corresponding section image. The ⁸⁹Zr radioactivity uptake in each ROI was calculated as the total intensity, photostimulated luminescence (PSL) per ROI area (mm²), corrected for the radioactive decay between injection and PI reading time points, normalized for differences in injected dose per gram of mouse weight and then background subtracted. To compare different image acquisitions, the ratios were calculated between the signals from the whole aorta of each animal and the whole aorta of the WT animal showing the lowest signal on each plate. Additionally, the ratios between the plaque areas in the aortic arch and nonplaque areas averaged over the whole aorta of each animal were calculated to evaluate plaque specificity.