1. Live imaging of M. abscessus infection<ol><li>Mount tricaine-anesthetized embryos in 1% low melting point agarose in a 35 mm petri dish prior to epifluorescence microscopy observations. Use a glass bottom dish for inverted confocal microscopy or a single cavity depression slide for upright confocal microscopy. Orient the embryo to the desired position and cover the solidified agarose with fish water containing tricaine.</li><li>Use an epifluorescence microscope with a 10 X objective for sequential fluorescence acquisition and transmission images of the entire embryo. Alternatively, use a confocal microscope with 40X or 63X objectives to visualize the activity of immune cells after an infection.</li></ol>

<table class='ck-table-resized'><tbody><tr><td>Low melting agarose</td><td>Sigma-Aldrich</td><td>&#160;</td><td>&#160;</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td>&#160;</td></tr></tbody></table>

live imaging of host-pathogen interactions in zebrafish embryos infected with mycobacterium abscessus

Source: <a href='https://app.jove.com/v/53130/deciphering-imaging-pathogenesis-cording-mycobacterium-abscessus'>Bernut, A., et al. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015)</a>This video demonstrates a protocol that uses transgenic zebrafish embryos expressing fluorescent macrophages and infected with fluorescent Mycobacterium abscessus rough strain to visualize host-pathogen interactions. It reveals how extracellular cording enables immune evasion, promoting bacterial survival and host cell damage.

Live Imaging of Host-Pathogen Interactions in Zebrafish Embryos Infected with Mycobacterium abscessus

Source: Bernut, A., et al. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos. J. Vis. Exp. (2015)This video ...

Begin with anesthetized, transparent, transgenic zebrafish embryos expressing fluorescently labeled macrophages.The embryos are pre-injected into the tail muscle with fluorescently labeled Mycobacterium abscessus rough strain to induce a localized infection.The absence of surface glycopeptidolipids in these bacteria promotes their aggregation.Immobilize the embryos in low-melting-point agarose and orient them to expose the infection site.Once the agarose solidifies, add fish water containing an anesthetic agent to maintain anesthesia and prevent dehydration.Monitor infection in real time via fluorescence microscopy.Following infection, endogenous macrophages migrate toward the bacteria and phagocytose individual cells.However, some bacteria form cords&#8212;elongated extracellular aggregates that replicate, spread, and resist phagocytosis, causing host cell death and immune infiltration.Despite this, immune cells fail to eliminate the cords, allowing bacteria and dead host cells to accumulate and form an abscess.This reveals cording as a key M. abscessus immune evasion strategy.For live imaging of M. abscessus infection, mount tricaine anesthetized embryos in 1% low-melting-point agarose in a 35-millimeter glass-bottom Petri dish for an inverted microscope or a single cavity depression slide for upright confocal microscopy. Orient the embryo to the desired position and cover the solidified agarose with fish water containing tricaine. Finally, use a fluorescence microscope with a 10X objective or a fluorescence confocal microscope with 40X or 63X objectives for sequential fluorescence acquisition and transmission imaging of the embryo.

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232 Views. Begin with anesthetized, transparent, transgenic zebrafish embryos expressing fluorescently labeled macrophages.The embryos are pre-injected into the tail muscle with fluorescently labeled Mycobacterium abscessus rough strain to induce a localized infection.The absence of surface glycopeptidolipids in these bacteria promotes their aggregation.Immobilize the embryos in low-melting-point agarose and orient them to expose the infection site.Once the agar...

Live Imaging of Host-Pathogen Interactions in Zebrafish Embryos Infected with Mycobacterium abscessus

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Low melting agarose	Sigma-Aldrich
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