A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae

Published: January 31, 2024

Abstract

Source: Thrikawala, S. et al., Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions. J. Vis. Exp. (2020)

The video demonstrates a zebrafish wounding and entrapment device technique for observing fluorescently labeled fungal pathogen infection in zebrafish larvae. Initial infection attracts macrophages, and as infection persists, neutrophil recruitment progresses at the injection site.

Protocol

1. Preparation of Aspergillus spores for injection From an Aspergillus spore suspension, calculate the volume needed to obtain 1 x 106 spores. The volume should be 20–100 μL; if not, produce a 10x dilution in 0.01% (v/v) sterile Tween-20 (Tween-water; Table of Materials). For example, if the calculated volume is 5 µL, produce a 10x dilution and use 50 µL of the diluted solution. NOT…

Declarações

The authors have nothing to disclose.

Materials

Dumont forceps #5 Roboz Surgical Instrument Co. RS-5045
Eyepiece reticle Microscope World RETR10 For calibrating needles, used in Stereomicroscope
Microinjector setup: Back pressure unit Applied Scientific Instrumentation BPU
Footswitch Applied Scientific Instrumentation FTSW
Micro pipet holder kit Applied Scientific Instrumentation M-Pip
Pressure injector Applied Scientific Instrumentation MPPI-3
Micromanipulator setup: Micromanipulator Narashige (Tritech) M-152
Magnetic stand and plate Tritech MINJ-HBMB
Needle puller Sutter Instrument P-97
Stereomicroscope Nikon SMZ-745
Tissuelyser II Qiagen 85300 To homogenize larvae
Material
Agarose Fisher BP160-500
Ampicillin sodium salt Fisher AAJ6380706
BSA, fraction V VWR AAJ65855-22
Kanamycin sulfate Fisher AAJ1792406
L spreaders Fisher 14 665 230
Microcapillary needles (no filament) World Precision Instruments (WPI) TW100-3
Microloader pipet tips VWR 89009-310 To load the needle with Aspergillus suspension
Miracloth VWR EM475855-1R To filter Aspergillus suspension
N-phenylthiourea Fisher AAL0669009 To prevent pigmentation
Phenol red, 1% solution Fisher 57254
Tricaine (Ethyl 3-aminobenzoate, methanesulfonic acid salt) Fisher AC118000500 To anesthetize larvae
Tween-20 Fisher BP337-500
Media and Solutions Components/Recipe
E3 media: 60x E3 17.2 g NaCl, 0.76 g KCl, 2.9 g CaCl2, 4.9 g MgSO4 · 7H2O, to 1 L with H2O
1x E3 16.7 ml 60x stock, 430 ul 0.05 M NaOH, to 1 L with H2O (optional: + 3 ml 0.01% methylene blue)
Tricaine stock solution 2 g Tricaine, 5 g Na2HPO4 · 7H2O, 4.2 ml 60X E3, to 500 ml with H2O, pH to 7.0-7.5 with NaOH
Glucose minimal media (GMM) agar: GMM agar 10 g Glucose (Dextrose), 50 ml 20x Nitrate salts, 1 ml TE, to 1 L with H2O, pH to 6.5 with NaOH, + 16 g Agar, autoclave
20x Nitrate salts 120 g NaNO3, 10.4 g KCl, 10.4 g, MgSO4 · 7H2O, 30.4 g, KH2PO4, to 1 L with H2O, autoclave
Trace elements (TE) 2.20 g ZnSO4 · 7H2O, 1.10 g H3BO3, 0.50 g MnCl2 · 4H2O, 0.16 g FeSO4 · 7H2O, 0.16 g CoCl2 · 6H2O, 0.16 g CuSO4 · 5H2O, 0.11 g (NH4)6Mo7O24 · 4H2O, 5.00 g Na2EDTA, to 100 ml with H2O, dissolve stirring overnight, autoclave

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A zWEDGI Technique to Visualize Fungal Pathogen-Infected Zebrafish Larvae. J. Vis. Exp. (Pending Publication), e21877, doi: (2024).

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