Mouse anti calprotectin

Immunohistochemistry was performed on 5 μm-thick FFPE tissue sections as previously indicated^{1 (link),39 (link)}. Tissues were stained with combinations of primary antibodies including polyclonal rabbit anti-IFNγ (Abcam, Cambridge, MA), rabbit anti-TNF (Bioss Antibodies, Boston, MA), rabbit anti-IL-4 (Abcam), rabbit anti-IL-10 (Abcam), rabbit anti-CD3 (Agilent, Santa Clara, CA), mouse anti-calprotectin (clone: MAC378, ThermoFisher), mouse anti-CD163 (clone: 1D6, ThermoFisher), and mouse anti-human alveolar macrophage antibody (HAM56, Enzo Life Sciences, Farmingdale, NY). Tissues were subsequently stained with appropriate anti-rabbit and anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 1 hr at room temp, and coverslips were mounted with DAPI-containing Prolong Gold mounting medium (ThermoFisher). Cells were imaged on an Olympus confocal microscope (Olympus, Waltham, MA) running FlowView 1000 software maintained by the University of Pittsburgh’s Department of Microbiology and Molecular Genetics, or a Nikon e1000 epifluorescence microscope running Nikon Elements (Nikon Instruments, Melville, NY). Images were annotated with Photoshop CS5.1 (Adobe Systems, San Jose, CA) and projections of z-stacks were made with the FIJI build of ImageJ^{42 (link)}. Quantitative image analysis was performed with CellProfiler^{43 (link)}.