Rhodamine red or fitc conjugated secondary antibody

The mouse aortas were harvested on days 7 or 14, embedded in OCT and sectioned. Cross sections of aortic tissues (9 µm) were fixed in 4% paraformaldehyde, blocked in 8% BSA in PBS and incubated with the following primary antibodies: rat anti-mouse MOMA-2 (1:200 dilution; Cat# ab33451, Abcam, Cambridge, United Kingdom), biotinylated-IL-12p40 (1:100 dilution; Cat# 505302, Biolegend, San Diego, CA), rat anti-mouse CD206 (1:200 dilution; Cat: MCA2235, Bio-Rad [Formerly AbD Serotec], Hercules, CA), or rabbit anti-mouse CD8a (1:200 dilution; Cat# bs-0648R, Bioss antibodies, Woburn, Massachusetts) followed by the appropriate rhodamine red- or FITC-conjugated secondary antibody (1:100–1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) or Streptavidin Alexa Fluor 488 conjugate (1:400 dilution; Cat# S-11223, Molecular Probes, Eugene, OR). Nuclei were counterstained with DAPI. Images were acquired on a Leica DM 2000 microscope fitted with a Leica DMC 4500 color camera and analyzed with Leica Application Suite (LAS) X software. Single- or double-color images were loaded into ImageJ and cell enumeration was performed in a blinded fashion. Data were obtained from 3–4 non-overlapping fields per aortic cross-section, 6–9 sections per aorta, n = 6–8 aortas per time point.

Cross sections (9 μm) of OCT-embedded frozen lung tissues were fixed in 4% paraformaldehyde, blocked in 8% BSA in PBS and incubated with the primary antibodies: anti-Histone H2B (1:100 dilution; Cat # SC-8651; Santa Cruz Biotechnology), anti-mouse MPO (1:100 dilution; Cat # HM1051BT; Hycult Biotech) followed by the appropriate rhodamine red- or FITC-conjugated secondary antibody (1:100–1:200; Jackson ImmunoResearch Laboratories). DNA was stained with DAPI. All images were acquired with QCapture software on a Nikon Eclipse microscope.