Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish
Trascrizione
Begin with a dish containing agarose-embedded, anesthetized, transgenic zebrafish larvae expressing the photoconvertible fluorescent protein in their leukocytes — phagocytic immune cells.
The left otic vesicle, pre-injected with red fluorescent dye-labeled bacteria, is positioned flat against the dish's bottom.
Within the otic vesicle, pre-injected bacteria cause a local infection, triggering immune cell migration and chemokine release, causing leukocyte infiltration into the otic vesicle.
Under a fluorescence microscope, observe the initial recruitment of green leukocytes and phagocytosis of red bacteria by leukocytes at the infection site.
Illuminate the otic vesicle with a 405-nanometer wavelength, irreversibly transforming the photoconvertible fluorescent protein from green to red and imparting red fluorescence to leukocytes — enabling their distinct visualization and precise tracking.
Now, examine the larvae at two different wavelengths at various depths, enabling visualization of both photoconverted red and remaining green fluorescence leukocytes.
Photoconverted red leukocytes disperse from the otic vesicles and scatter throughout the body, facilitating photoconversion-based leukocyte tracking.
