Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

Published: November 30, 2023

Abstract

Source: Harvie, E. A. et al., Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection. 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' migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

Protocol

1. Preparing Microinjection Needles 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. 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 …

Representative Results

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…

開示

The authors have nothing to disclose.

Materials

1.7 ml eppendorfs MidSci AVSS1700
14 ml falcon tube BD Falcon 352059
96 well plate Corning Incorporated 3596
Agar BD Biosciences 214030
CellTracker Red Molecular Probes, Invitrogen C34552
CNA agar Dot Scientific, Inc 7126A
Disposable transfer pipets Fisher Scientific 13-711-7m
Dissecting Scope Nikon SMZ745
DMSO Sigma Aldrich D2650
Fine tweezers Fine Science Tools 11251-20
Gel comb VWR 27372-482 4.2 mm width, 1.5 mm thick
Glass bottom dishes Custom made by drilling a 16–18 mm hole in the center of a 35-mm tissue culture dish bottom and placing a 22-mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light.
Glycerol Fisher Scientific G33-4
High melt agarose Denville Scientific, Inc. CA3510-6
Laser Scanning Confocal Microscope Olympus with FV-1000 system
Low melt agarose Fisher BP165-25
Magnetic stand Tritech (Narishige) GJ-1
Microinjection system Parker Picospritzer III
Microloader pipet tips Eppendorf 930001007
Micromanipulator Tritech (Narishige) M-152
Micropipette puller Sutter Instrument Company Flaming/Brown P-97
Nanodrop spectrophotmeter Thermo Scientific ND-1000
N-Phenylthiourea (PTU) Sigma aldrich P7629
Paraformaldheyde Electron Microscopy Sciences 15710
Petri Dishes Fisher Scientific FB0875712 100 mm x 15 mm
Phenol Red Ricca Chemoical Company 572516
Phosphate Buffered Saline Fisher Scientific BP665-1
Pronase Roche 165921
Protease peptone Fluka Biochemika 29185
Small cell culture dish Corning Incorporated 430165 35 mm x 10 mm
Thin wall glass capillary injection needles World Precision Instruments, Inc. TW100-3
Todd Hewitt Sigma Aldrich/Fluka Analytical T1438
Tricaine (ethyl 3-aminobenzoate) Argent Chemical Laboratory/Finquel C-FINQ-UE-100G
Yeast extract Fluka Biochemika 92144

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記事を引用
Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish. J. Vis. Exp. (Pending Publication), e21840, doi: (2023).

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