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.
Two-color Somite Fate Mapping in Skate Embryos
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.
Corresponding Organization :
Other organizations : University of Cambridge, Marine Biological Laboratory
Protocol cited in 2 other protocols
Variable analysis
- Location of injection (adjacent somites)
- Fate of the labeled somites over time (8-12 weeks)
- Temperature (15°C)
- Seawater flow-through system
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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