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…
Divulgazioni
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 |
Citazione di questo articolo
Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish. J. Vis. Exp. (Pending Publication), e21840, doi: (2023).