Two-color somite fate mapping experiments were performed as described in Criswell et al. (2017b) (link) and Ward et al. (2017) (link). S24 skate embryos were removed from their egg cases to a petri dish and anesthetized in tricaine (MS-222 1 mg/L in seawater). Adjacent somites were injected with the red-fluorescent lipophilic dye CM-DiI and the green-fluorescent lipophilic dye SpDiOC18 (ThermoFisher). Concentrated stocks of CM-DiI (5 µg/µL in absolute ethanol) and SpDiOC18 (2.23 µg/µL in dimethylformamide) were diluted 1:10 in 0.3 molar sucrose for injection. After injection embryos were returned to their egg cases and maintained in a flow-through seawater table at 15°C for 8–12 weeks post-injection.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
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