eoe sub articles

EoE Sub Articles

1. Preparing Microinjection Needles
<ol>
	<li>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.</li>
	<li>Using fine tweezers, break off the tip of the pulled needle so that the tip opening has a diameter of approximately 10 &#181;m.</li>
</ol>
2. Preparing Larval Injection ...

photoconversion-based leukocyte tracking in bacterial-infected transgenic zebrafish

Source: Harvie, E. A. et al., <a href="https://app.jove.com/v/52788">Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.</a>&#160;J. Vis. Exp. (2015)
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&#39; migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

<img alt="Figure 1" src="/files/ftp_upload/21840/21840_Figure1.jpg" /> 
Figure 1: Leukocyte recruitment to otic vesicle infection with&#160;S. iniae.&#160;(A) Neutrophil recruitment to&#160;S. iniae&#160;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...

Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

1. Preparing Microinjection Needles

	Prepare thin wall glass capillary injection needles (1.0 OD/0.75 ID) using a micropipette puller device with the ...

Begin with a dish containing agarose-embedded, anesthetized, transgenic zebrafish larvae expressing the photoconvertible fluorescent protein in their leukocytes &#8212; phagocytic immune cells.
The left otic vesicle, pre-injected with red fluorescent dye-labeled bacteria, is positioned flat against the dish&#39;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 &#8212; 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.