Borosilicate glass capillary needles

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.

Zebrafish embryos were harvested and incubated at 28 °C for the first 48 h. Al 48 hpf, embryos were anesthetized with 0.003% tricaine (Sigma). The human colorectal carcinoma cells with 80% confluence were trypsinized and harvested (approximately one million cells). Cells were labelled with Dil lipophilic dye and concentrated in 10 µL of phosphate-buffered saline (PBS) containing 2% polyvinylpyrrolidone 40 (PVP40) to prevent cell aggregation. They injected 200–300 cells in circulating embryos using a micromanipulador and an electric microijector IM-31 (Narishige). They were used to inject borosilicate glass capillary needles (1 mm O.D. × 0.75 mm I.D.; World Precision Instruments, Sarasota, FL, USA) with an outlet pressure of 34 kPa and an injection time of 30 ms. The embryos were incubated for four days after injection (hpi) al 34 °C in Petri dishes. At 24 hpi the embryos were imaged and the compound JHOR11 was added to the water. The concentration used was 80 µM. For in vivo monitoring of cells within the embryo, images were also taken at 96 hpi.

Prior to microinjections into Tg zebrafish embryos (fli1: EGFP) [84 (link)], HeLa tumor cells were labeled with the VybrantTM DiI dye (Invitrogen, ThermoFisher, Carlsbad, CA, USA). The fluorescent cells were loaded into borosilicate glass capillary needles (OD/ID: 1.00/0.75 mm, World Precision Instruments (WPI, Friedberg, Germany) and injected (200 cells), through a pneumatic picopump, into the Cuvier duct of 48 hpf embryos previously anesthetized with tricaine (0.02%, Sigma-Aldrich, Milan, Italy).
After injection, the xenograft-harboring larvae were incubated to recover at 34° C in fish water containing phenylthiourea (PTU) to inhibit the pigmentation process. After 2 h from the injection, the embryos were examined to ensure homogeneity of the xenografts. For drug treatments, only successfully injected xenograft larvae were selected, with approximately 200 HeLa fluorescent cells scattered around the caudal area.
The injected xenografts were exposed to the doses of 3d used above with DMSO as control.
For cancer cell imaging and fluorescence quantification, anesthetized embryos were distributed to 96-well plates with one embryo/well. Initially (time 0 h, pre-treatment) and after one-day post-treatment, the tumors were photographed with a Zeiss AxioObserver microscope for live-cell imaging.