Anopheles embryo fixation (approx. 2 hrs)
Protocol based on Goltsev 2004, modifications are in bold and are based on Juhn and James 2012, modifications by ACJ in italics
Before starting, prepare 1.3% bleach and beaker of dH2O, chill 50 mL conical tube of dH2O on ice, and put 100-250 mL beaker of dH2O on hot plate, but don’t turn it on yet.
Rinse embryos from oviposition material to mesh sieve.
To remove the exochorion, the embryos were incubated in 1.3% household bleach (check stock bottle to determine appropriate dilution) for 75 s. Pipette solution over embryos continuously. Immediately submerge mesh filter in dH2O when the 75s are up. Then wash thoroughly in deionized water. Blot the mesh dry with a paper towel. If it comes away pink, you have not completely washed away the bleach yet.
The embryos were then placed in glass scintillation vials with a 1:1 mixture of heptane and 4% formaldehyde in PBST, and then rocked gently on nutator for 25 min.
Afterwards, the formaldehyde phase was removed with a glass Pasteur pipette and replaced with RT deionized water. Invert gently 5x to wash. The water phase was then replaced once more, and the embryos were rocked gently an additional 30 min on the nutator. Turn on hot plate with dH20 to medium heat (~200-300 degrees).
The water phase was then removed, and scintillation vials were filled to the top with boiling deionized water (without removing the heptane phase) and incubated for 30 s.
The hot water phase was quickly removed and replaced with fresh deionized water prechilled on ice. Vials were then placed on ice for an additional 15 min.
The water phase was then completely removed, and the heptane phase was exchanged. To crack the endochorion, an equal volume of methanol was added, and the vials were strongly swirled once to break the clumps of embryos. Do not shake the embryos!
Vials containing the heptane–methanol mixture were allowed to stand for 10–15 min, and then the heptane and methanol phases were removed and the embryos were washed several times with methanol. The embryos can be stored in methanol at −20°C for several months.
The endochorions were manually peeled from the embryos using needles from 1-ml insulin syringes and toupee tape. Pipette embryos onto edges of a piece of toupee tape stuck to middle of a small plastic dish. Remove MeOH or allow it to dry briefly. Fill the dish with cold 70% EtOH. Then peel embryos. Be sure to nudge or brush them away from the tape and into the EtOH or they will become permanently stuck to the tape. If the embryos are not sticking well, or if they are too sticky, you can add dH2O or 100% EtOH to adjust. Pipette peeled embryos with p200 tip with end cut off and transfer to 1.5 mL tube. Once all embryos are collected, they can be rinsed in 100% MeOH a few times and then stored at -20 in MeOH until use.
Protocol based on Goltsev 2004, modifications are in bold and are based on Juhn and James 2012, modifications by ACJ in italics
Before starting, prepare 1.3% bleach and beaker of dH2O, chill 50 mL conical tube of dH2O on ice, and put 100-250 mL beaker of dH2O on hot plate, but don’t turn it on yet.
Rinse embryos from oviposition material to mesh sieve.
To remove the exochorion, the embryos were incubated in 1.3% household bleach (check stock bottle to determine appropriate dilution) for 75 s. Pipette solution over embryos continuously. Immediately submerge mesh filter in dH2O when the 75s are up. Then wash thoroughly in deionized water. Blot the mesh dry with a paper towel. If it comes away pink, you have not completely washed away the bleach yet.
The embryos were then placed in glass scintillation vials with a 1:1 mixture of heptane and 4% formaldehyde in PBST, and then rocked gently on nutator for 25 min.
Afterwards, the formaldehyde phase was removed with a glass Pasteur pipette and replaced with RT deionized water. Invert gently 5x to wash. The water phase was then replaced once more, and the embryos were rocked gently an additional 30 min on the nutator. Turn on hot plate with dH20 to medium heat (~200-300 degrees).
The water phase was then removed, and scintillation vials were filled to the top with boiling deionized water (without removing the heptane phase) and incubated for 30 s.
The hot water phase was quickly removed and replaced with fresh deionized water prechilled on ice. Vials were then placed on ice for an additional 15 min.
The water phase was then completely removed, and the heptane phase was exchanged. To crack the endochorion, an equal volume of methanol was added, and the vials were strongly swirled once to break the clumps of embryos. Do not shake the embryos!
Vials containing the heptane–methanol mixture were allowed to stand for 10–15 min, and then the heptane and methanol phases were removed and the embryos were washed several times with methanol. The embryos can be stored in methanol at −20°C for several months.
The endochorions were manually peeled from the embryos using needles from 1-ml insulin syringes and toupee tape. Pipette embryos onto edges of a piece of toupee tape stuck to middle of a small plastic dish. Remove MeOH or allow it to dry briefly. Fill the dish with cold 70% EtOH. Then peel embryos. Be sure to nudge or brush them away from the tape and into the EtOH or they will become permanently stuck to the tape. If the embryos are not sticking well, or if they are too sticky, you can add dH2O or 100% EtOH to adjust. Pipette peeled embryos with p200 tip with end cut off and transfer to 1.5 mL tube. Once all embryos are collected, they can be rinsed in 100% MeOH a few times and then stored at -20 in MeOH until use.