Immunohistochemistry Protocol for IHC

For IHC ^{75 (link)}, 5 µm tissue sections were cut, mounted, dewaxed and rehydrated and then treated as described in Table S4. Epitope retrieval (15 min at 98–100°C) was followed by blocking (TBS+0.5% casein; 10 min), rinsing [TBS+0.05% Tween-20 (TBS-Tw)] and an incubation with primary antibody (60 min). Samples were washed in TBS-Tw and blocked using 1.5% H₂O₂ in TBS-Tw (5 min) (peroxidase block was not performed for MPO-IHC). Horseradish peroxidase-conjugated secondary antibodies (Table S4) were detected using ImmPact-DAB substrate (Vector Labs). All slides were washed in distilled water, counterstained with hematoxylin, mounted with Clear Mount (Invitrogen), dried for 60 min at 40°C and mounted with Permount (Electron Microscopy Sciences).