After dewaxing and rehydration, tissue sections were blocked with 0.3% BSA in Dulbecco’s PBS (DPBS) for 30 minutes at room temperature in a hydration chamber and then incubated overnight with the prepared antibody cocktail (Supplemental Table 3, Netrin-1-152Sm, CD11b-149Sm, F4/80-159Tb, and HistoneH3-176Yb [to identify nuclei]) in 100 μL DPBS at 4°C in a hydration chamber. The next day, slides were washed, and sections were stained with Ir-Intercalator in DPBS for 60 minutes at room temperature in a hydration chamber, then washed with TBS-T for 5 minutes (twice), then washed with Milli-Q water for 5 minutes (twice), and then allowed to air-dry for more than 30 minutes at room temperature. Slides were imaged with the Hyperion Imaging Mass Cytometer (27). Data were analyzed using Histology Topography Cytometry Analysis Toolbox (HistoCAT) (59). The fold change in cell density per mm2 was calculated and compared between groups.

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