Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

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…

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