Characterization of Macrophage Extracellular Traps in Mouse Lung Tissue Samples

Macrophage extracellular traps, METs, are web-like structures released by macrophages to trap and eliminate pathogens.

METs comprise extracellular chromatin fibers associated with matrix metalloproteinase-9, MMP-9, and citrullinated histone H3, H3Cit — a modified histone protein in which the arginine is converted into citrulline.

To characterize METs in formalin-fixed, paraffin-embedded murine lung tissue sections, firstly, dry the slides. Immerse in xylene to deparaffinize the sections. Use aqueous ethanol to rehydrate the sections.

Heat the sections to break the formalin-induced protein crosslinks, exposing the target antigens for subsequent antibody binding.

Add a protein-containing solution to block non-specific binding sites. Incubate with a cocktail of anti-MMP-9, anti-H3Cit, and anti-F4/80 primary antibodies.

The anti-F4/80 antibody binds to the macrophage cell surface glycoprotein F4/80. The anti-MMP-9 and anti-H3Cit antibodies bind to the MMP-9 and H3Cit in METs respectively. Add different fluorophore-conjugated secondary antibodies that bind to their respective primary antibodies bound to their targets.

Mount the sections with DAPI-containing mounting medium. DAPI stains the METs' chromatin fibers. Using a confocal microscope, image the sections to visualize the fluorescence of the MET markers.

METs are characterized by their extracellular chromatin fibers and the co-expression of F4/80, MMP-9, and H3Cit.