Axiozoom v16 fluorescence microscope

Mice were deeply anesthetized with isoflurane and transcardially perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 1x PBS. Whole brains were removed and post-fixed in 4% PFA overnight at 4 °C and subsequently transferred to 1x PBS for storage at 4 °C until further processing. Coronal brain sections (50 µm thick) were collected in 1x PBS using a Leica VT1200 vibratome (Leica Biosystems GmBH, Wetzlar, Germany). Sections were mounted with DAPI-Fluoromount-G aqueous mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA) onto Superfrost Plus glass slides (VWR International, Radnor, PA, USA). Images were taken with an AxioZoom.V16 fluorescence microscope (Carl Zeiss Microscopy LLC, Thornwood, NY, USA).

For osteogenic differentiation, mouse limb subpopulations and total cells were directly sorted at 30,000 cells/cm² in 96 well plates to reach the 80% confluence. Cells for each condition were incubated for 15 days in 1 mL of Complete MesenCult Osteogenic Medium (StemCell Technologies, Vancouver, BC, Canada). Debris or detached cells were washed with PBS, and the medium was replaced with fresh medium every 3–4 days. After 15 days of induction, differentiated cells were washed with PBS and fixed in 4% paraformaldehyde for 30 min at 4°C. For alizarin red staining, cells were fixed in 10% of formaldehyde-PBS for 10 min at room temperature. After washing, cells were stained with 0.2% Alizarin S-Red solution (pH 4.2) for 20 min. Excess of alizarin staining was washed twice with water for further image acquisition.
Chondrogenic differentiation was evaluated by micromass assays. Freshly isolated subpopulations and total cells were directly seeded in 48 well plates at 3 × 10⁵ cells in 10 μL of DMEM-HG medium (Life Technologies), supplemented with 10% FBS (Life Technologies). After permit cell attachment for 2 h, micromass was flooded in DMEM-HG medium (Life Technologies). Cultures were maintained for 3 days under a 5% CO₂ atmosphere until Alcian blue staining. Chondrogenic and osteogenic differentiation images were acquired with an AxioZoom V.16 fluorescence microscope (Carl Zeiss).

Melanomas were generated by Transgene Electroporation in Adult Zebrafish as previously described^{13 (link),21 (link)}. To generate BRAF^V600E p53^−/− PTEN ^ko melanomas, adult (3–6 months) transgenic zebrafish (casper (mitfa^−/−;mpv17^−/−);mitfa:BRAF^V600E;p53^−/−) were injected with the following tumor initiating plasmids: MinicoopR-tdT (250 ng/µL), mitfa:Cas9 (250 ng/µL), U6:sgptena (23 ng/µL), U6:sgptenb (23 ng/µL) and Tol2 (57 ng/µL). For dgat1a knockout experiments, zebrafish were injected with an additional guide plasmid of U6:sgNT (23 ng/µL) or U6:sgdgat1a (23 ng/µL). Adult male and female zebrafish were anesthetized in tricaine and injected with 1 µL of tumor-initiating plasmids below the dorsal fin, electroporated using the CM 830 Electro Square Porator from BTX Harvard Apparatus, and recovered in fresh water. For dgat1a knockout experiments, zebrafish were imaged every 2 weeks using brightfield and fluorescence imaging using a Zeiss AxioZoom V16 fluorescence microscope. To quantify tumor area, images were analyzed in MATLAB R2020a by quantifying pixels positive for melanin and tdTomato.

Zebrafish (Danio rerio) were reared under standard conditions in a 14 h/10 h light cycle according to the guidelines of the local authorities (Magistratsabteilung MA58 of the municipal administration of Vienna, Austria) under licenses GZ:565304-2014-6 and GZ:534619-2014-4. For xenotransplantation experiments, the pigment mutant strain mitfa^b692/b692; ednrba^b140/b140 was used. mitfa^b692/b692; ednrba^b140/b140 embryos raised at 28 °C were anaesthetised with Tricaine (0.16 g/l Tricaine (Cat# E1052110G, Sigma-Aldrich), adjusted to pH 7 with 1 M Tris pH 9.5, in E3) and xenotransplanted at 2 days post fertilisation (dpf)^{107 (link)}. For xenotransplantation, a micromanipulator (Cat# M3301R, World Precision Instruments) holding a borosilicate glass capillary (Cat# GB100T-8P, without filament, Science Products) connected to a microinjector (FemtoJet 4i, Eppendorf) was used. Transplantation capillaries were pulled with a needle puller (P-97, Sutter Instruments) and loaded with approximately 5 µl of tumour cell suspension. Cells were injected into the perivitelline space (PVS) of larvae. Visual inspection was carried out at 2 h post-injection on an Axio Zoom.V16 fluorescence microscope (Zeiss, Jena) and only correctly injected larvae were used in subsequent experiments and further maintained at 34 °C.