CS
Celia E Shiau
  • University of North Carolina at Chapel Hill Chapel Hill
Research fields
  • Plant science
Brain-localized and Intravenous Microinjections in the Larval Zebrafish to Assess Innate Immune Response
Authors:  Alison M. Rojas and Celia E. Shiau, date: 04/05/2021, view: 4801, Q&A: 0

Creating a robust and controlled infection model is imperative for studying the innate immune response. Leveraging the particular strengths of the zebrafish model system, such as optical transparency, ex utero development, and large clutch size, allows for the development of methods that yield consistent and reproducible results. We created a robust model for activation of innate immunity by microinjecting bacterial particles or live bacteria into larval zebrafish, unlike previous studies which largely restricted such manipulations to embryonic stages of zebrafish. The ability to introduce stimuli locally or systemically at larval stages provides significant advantages to examine host response in more mature tissues as well as the possibility to interrogate adaptive immunity at older larval stages. This protocol describes two distinct modes of microinjection to introduce lipopolysaccharide (LPS) or bacteria into the living larval zebrafish: one localized to the brain, and another into the bloodstream via the caudal vein plexus.


Graphic abstract:



Schematic shows the two distinct modes of larval zebrafish microinjection, either in the brain parenchyma or in the blood stream intravenously. Reagents introduced into the zebrafish to assess immune response are depicted in the “injection components” as described in the protocol.

Liposomal Clodronate-mediated Macrophage Depletion in the Zebrafish Model
Authors:  Linlin Yang, Alison M. Rojas and Celia E. Shiau, date: 03/20/2021, view: 4874, Q&A: 0

The ability to conduct in vivo macrophage-specific depletion remains an effective means to uncover functions of macrophages in a wide range of physiological contexts. Compared to the murine model, zebrafish offer superior imaging capabilities due to their optical transparency starting from a single-cell stage to throughout larval development. These qualities become important for in vivo cell specific depletions so that the elimination of the targeted cells can be tracked and validated in real time through microscopy. Multiple methods to deplete macrophages in zebrafish are available, including genetic (such as an irf8 knockout), chemogenetic (such as the nitroreductase/metronidazole system), and toxin-based depletions (such as using clodronate liposomes). The use of clodronate-containing liposomes to induce macrophage apoptosis after phagocytosing the liposomes is effective in depleting macrophages as well as testing their ability to phagocytose. Here we describe a detailed protocol for the systemic depletion of macrophages in zebrafish larvae by intravenous injection of liposomal clodronate supplemented with fluorescent dextran conjugates. Co-injection with the fluorescent dextran allows tracking of macrophage depletion in real time starting with verifying the successful intravenous injection to macrophage uptake of molecules and their eventual death. To verify a high degree of macrophage depletion, the level of brain macrophage (microglia) elimination can be determined by a rapid neutral red vital dye staining when clodronate injection is performed at early larval stages.


Graphical abstract:



Experimental workflow for in vivo macrophage-specific depletion by liposomal clodronate in larval zebrafish


We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.