Fluorescence Imaging of Biomaterial-Associated Bacterial Infection in Zebrafish Embryos

Abstract

Source: Zhang, X., et al. A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection. J. Vis. Exp. (2019)

This video demonstrates fluorescence imaging of zebrafish embryos injected with Staphylococcus aureus, enabling quantification of infection progression and bacterial distribution to assess the impact of biomaterial microspheres on host-pathogen interactions in vivo.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Fluorescence Microscopy of Infection Progression and Provoked Cell Infiltration in Zebrafish Embryos

1. Anaesthetize the embryos by placing them in a 100 mm Petri dish containing 0.02% (w/v) 3-aminobenzoic acid (Tricaine) to anaesthetize them. After 5 min, transfer the embryos to the agarose plate overlaid with E3 medium containing 0.02% (w/v) Tricaine and align them in one orientation for injection. Pipette 500 µL of a PBS (phosphate buffered saline)-2% (wt) methylcellulose solution into a Petri dish containing E3 medium with 0.02% (w/v) Tricaine. Place the embryos in the methylcellulose solution and keep them straight and horizontal.
   NOTE: Methylcellulose solution is used as a “glue”, which due to its viscosity, can temporarily immobilize embryos in best orientation for imaging.
2. Use a stereo fluorescence microscope equipped with bright field, mCherry, green fluorescent protein (GFP), and UV (ultraviolet) filters to image individual embryos under identical optimized settings (e.g., intensity, gain, and exposure time) at 160x magnification. Set the focal plane such that the tissue damage caused by the injection is in focus, using the bright field filter. Set the Z-stack depth at 10 µm and step size at 5 µm, which allows for the recording of 3 consecutive images.
3. Image individual embryos once daily from 5 h post-injection until 2 days post-injection. Exclude dead embryos (no heartbeat or embryo partly degraded) for further imaging and analysis. Maintain embryos individually in E3 medium in 48-well plates. Refresh the medium daily.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 µm diameter polystyrene microspheres (blue fluorescent)	Life technology/ThemoFisher	F8829
3-aminobenzoic acid (Tricaine)	Sigma-Aldrich	E10521-50G
Stereo fluorescent microscope LM80	Leica	MDG3610450126
Methyl cellulose 4000cp	Sigma-Aldrich	MO512-250G
Petri-dish	Falcon	353003
Petri-dish	Biomerieux	NL-132
ImageJ	Not applicable	Not applicable	link: https://imagej.nih.gov/ij/download.html
GraphPad 7.0	Prism	Not applicable