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Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes' migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

1. Preparing Microinjection Needles

  1. Prepare thin wall glass capillary injection needles (1.0 OD/0.75 ID) using a micropipette puller device with the following settings: air pressure 200, heat 502, pull 90, velocity 80, time 70, air time at the start of pull 5, air time at end of pull 5.
  2. Using fine tweezers, break off the tip of the pulled needle so that the tip opening has a diameter of approximately 10 µm.

2. Preparing Larval Injection .......

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Figure 1
Figure 1: Leukocyte recruitment to otic vesicle infection with S. iniae(A) Neutrophil recruitment to S. iniae infection. (i) Successful injection of a phenol red-labeled inoculum into the otic vesicle. (ii-iv) Sudan Black staining of larvae for investigation of neutrophil recruitment at 2 h.......

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