Encyclopedia of Experiments: Immunology
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Transcript
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.
