Pc 10 micropipette puller

A. baerii prolarvae were anesthetized with using 200 mg/L concentration of MS-222 and placed on a 1% agarose bed. Microinjection needles of 20 μm diameter were prepared from glass capillaries (World Precision Instruments, Sarasota, FL, USA) with a PC-10 Micropipette puller (Narishige, Tokyo, Japan) and EG-401 Micro grinder (Narishige). Microinjection was performed manually with a M-152 micromanipulator (Narishige). Injections were made at the border region between the prolarval body and the yolk extension [15 (link)]. The injection was traced using a visible methylene blue staining dye (Sigma-Aldrich) at 0.5 mg/mL. The amount of dye solution injected into each prolarva was estimated to be approximately 15–20 nL depending on injection batches. A group of 7–9 prolarvae was anesthetized for each trial, with eight replicate trials made to yield a dataset of 60 microinjected prolarvae. These trials were carried out at three different age stages (Day 0 to Day 1, Day 2, and Day 3). After injection, prolarvae were transferred to the recovery tank and then to rearing cages for monitoring post-procedure viability for up to 120 h post injection, at 12-h intervals. Two control groups, including an anesthetized but not-injected group, and a non-anesthetized group were also prepared on each day of treatment.

Fluorescently labelled cells (with or without BCG) were subcutaneously injected into the PVS of 48 h old zebrafish embryos using a micromanipulator. For this, embryos were mechanically dechorionated using micro-surgical forceps. Stained cell suspensions were centrifuged for 5 min at 250× g and resuspended in PBS to reach a cell concentration of ~300 cells/nl. Borosilicate glass capillaries (World-Precision instruments, Sarasota, FL, USA, #TW100-4) were pulled using a Narishige (Setagaya City, Tokyo, Japan) PC-10 micropipette puller and filled with 3–5 µL of cell suspension. At the needle tip, a needle opening was created using fine forceps and the droplet size was calibrated. Dechorionated embryos were put on pre-warmed 2% agarose plates and anesthetized with 1 mg/mL Tricaine before microinjecting the tumor cells. Embryos were transferred back to the E3 medium supplemented with PTU following injection.
Using fluorescence microscopy, successfully injected embryos, which (for PVS injections) showed tumor cells in the PVS but not in the yolk or in the bloodstream, were selected. The selected zebrafish tumor xenografts were incubated at 35.5 °C in 24-well plates containing the E3 medium supplemented with PTU until 3 dpi.

Microelectrodes were fabricated according to a procedure described previously.^{79 (link)} Simply, 25 μm or 100 μm diameter wires of Pt (Mint of Poland), Au, or Cu (Alfa Aesar) were inserted in a borosilicate glass capillary. Then they were mounted into a PC-10 micropipette puller (Narishige) to melt the glass and to seal the wire tightly inside the capillary under vacuum conditions to avoid gas bubbles. The end of the microelectrode was polished with P2000 grit silicon carbide sand paper.