Borosilicate glass capillary needles

Zebrafish and embryos were raised, staged and maintained according to standard procedures. The Institutional Committee for Animal Welfare of the Leiden University Medical Center (LUMC) approved this study. Tg(Fli1:GFP) zebrafish embryos were dechorionated at two days post-fertilisation (dpf). Single cell suspensions of melanoma cells were prepared in PBS and kept at 4°C before implantation. The cell suspension was loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus) and the injections were performed using a Pneumatic Picopump and a manipulator (World Precision Instruments, Stevenage, UK). Dechorionated embryos were anaesthetised with 0.003% 3-amino benzoic acid ethyl estertricaine, (Sigma)] and mounted on 10 cm Petridishes coated with 1% agarose. Approximately 200 cells were injected at the duct of Cuvier (DoC). Implanted zebrafish embryos were maintained at 33°C. Zebrafish in the Non-Silencing (NS) + SB431542 group were treated with 1μM SB-431542 added to the eggwater. All implantations were repeated at least three times with at least 30 embryos per group.

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.

After fertilization (d = 0) embryos were positioned on a 10 cm Petri dish coated with 1% agarose and injected with 1 nl 0.8 M D-2-HG, L-2-HG or PBS during the 2–8 cell stages of development [41 (link)]. The compounds were loaded into borosilicate glass capillary needles (1.0 mm OD × 0.78 mm ID × 100 mm L; Harvard Apparatus) and the injections were performed using a Pneumatic Pico pump. Per experiment approximately 20 zebrafish embryos per group were used. Experiments with PBS and D-2-HG conditions were performed twice at different time points. At 8 days post fertilization (dpf) water was removed and 4% paraformaldehyde was added to fix the zebrafish embryos. Double staining with alizarin red and alcian blue was performed as described elsewhere [42 (link)]. Alizarin Red positive vertebrate rings were counted to quantify bone development. To determine statistical significance an independent sample t-test was performed using SPSS v20.

The transgenic fish line Tg(fli1:GFP) was used [16 (link), 17 (link)]. The tumor cell lines were fluorescently labelled with mCherry using plv-mCherry lentiviruses. G418 was used as selection marker. Embryo preparation and tumor cell implantation was done as previously described [18 ]. Briefly, Tg(fli1:GFP) zebrafish embryos were dechorionated 2 days post fertilization. Single cell suspensions of 66cl4 wildtype cells and variants were re-suspended in PBS and kept at 4 °C until transplantation. Cell suspensions were loaded into borosilicate glass capillary needles (1 mm O.D. × 0.78 mm I.D.; Harvard Apparatus) and injected into the duct of Cuvier (DoC) using a Pneumatic Picopump and a manipulator (WPI, Stevenage, UK). After injection of approximately 400 cells, zebrafish embryos were maintained at 33 °C. Invasive clusters were quantified 6 days post-implantation (dpi). Zebrafish embryos were fixated with 4% paraformaldehyde at 4 °C overnight and were imaged in PBS using a Leica SP5 STED confocal microscope. Confocal stacks were processed for maximum intensity projections with Image J. Brightness and contrast of images were adjusted with Adobe Photoshop CS6. For each cell line, at least fifty embryos were analyzed.