Zebrafish embryos were anesthetized with 0.3 mg/mL tricaine at 48 h post-fertilization (hpf) before injection. Each vector was loaded into borosilicate glass capillary needles, and the injections were performed using an IM-31 electric microinjector (Narishige). The established parameters’ pressure was 30–35 kPa, with an injection time of 20–30 ms. Injections were executed manually into the midbrain area between the telencephalon and the rhombencephalon, as shown in Figure 1 A. The injected volume ranged from 1 to 2 nL.
Im 31 electric microinjector
Manufactured by Narishige
Sourced in United Kingdom
The IM-31 Electric Microinjector is a laboratory device designed for precision microinjection applications. It provides accurate and controlled injection of small volumes of liquids or gases into biological samples, such as cells or tissues. The core function of the IM-31 is to deliver measured quantities of fluids with high repeatability and consistency, enabling researchers to perform delicate micromanipulation tasks.
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5 protocols using im 31 electric microinjector
1
Zebrafish Embryo Microinjection Protocol
2
Zebrafish Xenotransplantation via Yolk Injection
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Two days post fertilization (dpf), zebrafish embryos were dechorionated (if needed) and anesthetized with 0.003% tricaine (Sigma). Cells were suspended at 10,000-20,000 cells/μl in complete McCoy and maintained at room temperature for no longer than 2 h before they were injected. The cell suspension was loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus), and injections were performed using IM-31 Electric Microinjector (Narishige) with an output pressure of 34 kPa and 30 ms injection time. The injections were performed manually right into the yolk of the embryo. Incorrectly injected embryos without cells inside of the yolk, or showing them in the circulation after xenotransplantation were discarded.
3
Xenograft Breast Cancer Assay
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Embryos were collected and grown at 28 °C in Petri dishes at a ratio of 50 embryos/plate. Two days post fertilization (dpf), embryos were dechorionized (if needed) and anesthetized with 0.003% tricaine (Sigma). GFP-labeled MCF7 breast cancer cells (10,000–20,000 cells/µL) were loaded into borosilicate glass capillary needles (1 mm O.D. × 0.75 mm I.D.; World Precision Instruments—Hitchin, Hertfordshire, UK) and injected at an average of 150–250 cells/embryo into the yolk sac using IM-31 electric microinjector (Narishige). We discarded embryos showing cells outside the yolk.
4
Toxicity Assay of Nanoparticles in Zebrafish
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Toxicity assay by injection was carried out in 48 hpf embryos without chorion. Embryos were anesthetized with 0.003% tricaine (CAS 886–86-2) from Sigma and injected using a borosilicate glass capillary needle (1 mm O.D. × 0.58 mm I.D.; Harvard apparatus) controlled with IM-31 Electric Microinjector (Narishige) with an output pressure of 34 kPa and 25 ms injection time. Five µg/mL of ET-NEs and C-NEs were injected in the yolk or the caudal vein. The embryos were incubated at 28 ºC and evaluated under inverted optical microscope at 24, 48, 72 and 96 h post-injection (hpi) in order to analyze development alterations, malformations, and mortality. Three replicates of 20 embryos were used for each experiment.
5
Zebrafish Xenograft Tumor Model
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Zebrafish embryos were collected and incubated at 28.5 °C during the first 48 h hpf. At 48 hpf the embryos were anaesthetized with 0.003% tricaine (Sigma). MM96L human melanoma cells were cultured at 37 °C and 5% CO2 until they reached ~70% confluence. MM96L cells were then trypsinized and one million cells were harvested, labeled with Dil lipophilic dye and concentrated in 10 µL of phosphate-buffered saline (PBS) containing 2% polyvinyl-pyrrolidone 40 (PVP40) to avoid cell aggregation. Borosilicate glass capillary needles (1 mm O.D. × 0.75 mm I.D.; World Precision Instruments) were used to inject 200–300 cells into the circulation of the embryos using a micromanipulator and a IM-31 Electric Microinjector (Narishige) with an output pressure of 34 kPa and 30 ms injection time. After the injection, embryos were incubated for three days post-injection (hpi) at 34 °C in 30-mL Petri dishes containing SDTW.
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