D. maculatus Embryo Fixation, Jie Xiang
1. Prepare a collecting basket with a small piece of mesh in the center.
2. Transfer appropriately staged embryos to the basket.
3. Treat embryos with 50% bleach for four minutes. Stir occasionally.
4. Rinse with tap water three times and embryo wash buffer once (8g NaCl, 500 µl of Triton X-100 in 2L dH2O). Make sure to wash all embryos off the wall of the vial to the center of the mesh.
5. Transfer the mesh to a 1.5 ml Eppendorf tube with 1 ml of dH2O using forcep. (Note: use ~ 200 µl of embryos in each tube. If there are too many embryos, split them to more tubes)
6. Remove the mesh once most embryos are off the mesh.
7. Submerge the tube with embryos in boiling water for 3 minutes.
8. Transfer the tube to ice immediately and let it stay on ice for 7 minutes.
9. Remove dH2O without losing embryos.
10. Add 500 µl of heptane and 500 µl of 4% PFA (paraformaldehyde).
11. Fix the embryos on the shaker at high speed (250 rpm) for 20 minutes.
12. Remove the aqueous phase (PFA, lower layer) without sucking up embryos.
13. Add 800 µl of 100% MeOH. Cap the tube and shake it vigorously for 20 seconds.
14. Flick the tube gently to let the embryos sink to the bottom. Note: If most embryos are still floating around, shake the tube for another 20 seconds.
15. Remove as much solution as possible without losing any embryos.
16. Rinse twice with 800 µl of 100% MeOH. Make sure to rinse embryos off the wall of the vial.
17. Add 800 µl of 100% MeOH and store the tube at -20° C.
The embryos will be dissected in PBST to remove the eggshell before in situ hybridization or antibody staining.
1. Prepare a collecting basket with a small piece of mesh in the center.
2. Transfer appropriately staged embryos to the basket.
3. Treat embryos with 50% bleach for four minutes. Stir occasionally.
4. Rinse with tap water three times and embryo wash buffer once (8g NaCl, 500 µl of Triton X-100 in 2L dH2O). Make sure to wash all embryos off the wall of the vial to the center of the mesh.
5. Transfer the mesh to a 1.5 ml Eppendorf tube with 1 ml of dH2O using forcep. (Note: use ~ 200 µl of embryos in each tube. If there are too many embryos, split them to more tubes)
6. Remove the mesh once most embryos are off the mesh.
7. Submerge the tube with embryos in boiling water for 3 minutes.
8. Transfer the tube to ice immediately and let it stay on ice for 7 minutes.
9. Remove dH2O without losing embryos.
10. Add 500 µl of heptane and 500 µl of 4% PFA (paraformaldehyde).
11. Fix the embryos on the shaker at high speed (250 rpm) for 20 minutes.
12. Remove the aqueous phase (PFA, lower layer) without sucking up embryos.
13. Add 800 µl of 100% MeOH. Cap the tube and shake it vigorously for 20 seconds.
14. Flick the tube gently to let the embryos sink to the bottom. Note: If most embryos are still floating around, shake the tube for another 20 seconds.
15. Remove as much solution as possible without losing any embryos.
16. Rinse twice with 800 µl of 100% MeOH. Make sure to rinse embryos off the wall of the vial.
17. Add 800 µl of 100% MeOH and store the tube at -20° C.
The embryos will be dissected in PBST to remove the eggshell before in situ hybridization or antibody staining.