Harlequin bug, Murgantia histrionica, Embryo Fixation, Jessica Hernandez
Fixing too many embryos in one tube is not ideal, 20-25 embryos works best (use 2mL tubes).
Fixing too many embryos in one tube is not ideal, 20-25 embryos works best (use 2mL tubes).
- Bring 300 mL of water in large beaker to a boil. Place a small beaker with 5ml ddH2O water on hot plate and bring to a boil (volume used here will depend on how many tubes you have). Take PFA out of the freezer and let it melt. Make sure that everything in the PFA tube is dissolved.
- Add 600 ul of hot ddH2O to tubes with embryos and immediately place tubes in the boiling water. Try to do this fast. Boil for 3 mins
- Remove embryos and place on ice for 10-15 mins, this helps when removing caps.
- Remove the water and add 1000 ul of PBST.
- Under the microscope remove the embryo’s cap. When doing this try to be careful as the embryos can be damaged easily, try not to squeeze the embryos with the forceps.
6. Once cap is removed, add 600ul 12% PFA and 600 ul Heptane
7. Shake for 20 mins at about 200 RPM, avoid shaking them at maximum speed as such can damage the embryos.
8. Remove the bottom layer (PFA layer), add 600 ul of 100 % MeOH
9. Remove Heptane:MeOH and rinse 3x with 100% MeOH
10. Add 500 ul of MeOH and 500 ul of Xylene. Let rock at RT for 30 mins to 1 hour. This should make the shell of the embryos softer and easier to dissect, such will not remove the shell.
11. Remove MeOH:Xylene and rinse 3x with 100 % MeOH
12. Store in 1000ul MeOH at -20C.
Embryo Dissection and In-situ ready embryos
50% MeOH
25% MeOH
Rinse 2x with PBST
2. Dissect embryos under the microscope with forceps carefully.
7. Shake for 20 mins at about 200 RPM, avoid shaking them at maximum speed as such can damage the embryos.
8. Remove the bottom layer (PFA layer), add 600 ul of 100 % MeOH
9. Remove Heptane:MeOH and rinse 3x with 100% MeOH
10. Add 500 ul of MeOH and 500 ul of Xylene. Let rock at RT for 30 mins to 1 hour. This should make the shell of the embryos softer and easier to dissect, such will not remove the shell.
11. Remove MeOH:Xylene and rinse 3x with 100 % MeOH
12. Store in 1000ul MeOH at -20C.
Embryo Dissection and In-situ ready embryos
- Rehydrate embryos in MeOH gradient, 2 mins in each solution
50% MeOH
25% MeOH
Rinse 2x with PBST
2. Dissect embryos under the microscope with forceps carefully.
3. Rinse embryos and invert for 10 seconds in each solution (1mL):
25% MeOH
50% MeOH
75% MeOH
2x 100% MeOH
Remove 500uL MeOH and add 500uL heptane, invert tube for 10 seconds. This is necessary to remove a second membrane. Embryos will float indicating that they have this membrane, they should stop floating when the membrane comes off.
4. Rehydrate embryos to PBST
50% MeOH
25% MeOH
2X PBST
5. Add 1mL 4%PFA and fix for one hour. You can also use 8% PFA, this is helpful when doing nuclear stain.
Proceed to In-situ Protocol.
25% MeOH
50% MeOH
75% MeOH
2x 100% MeOH
Remove 500uL MeOH and add 500uL heptane, invert tube for 10 seconds. This is necessary to remove a second membrane. Embryos will float indicating that they have this membrane, they should stop floating when the membrane comes off.
4. Rehydrate embryos to PBST
50% MeOH
25% MeOH
2X PBST
5. Add 1mL 4%PFA and fix for one hour. You can also use 8% PFA, this is helpful when doing nuclear stain.
Proceed to In-situ Protocol.