Updated 7/25/22 by Patricia Graham
Tribolium embryo fixation
1) Collect embryos in a 300um sieve.
a. Pour onto a blank piece of paper.
b. Tap onto a second piece – leaving bits of bug and flour behind.
c. Repeat tapping until just eggs are left.
d. Transfer them to a basket.
2) Rinse well with water to remove flour on embryos.
3) Dechorionate in a 1:1 mix of household bleach and dH2O for 3 minutes.
4) Rinse well with water.
5) Fix in 1:1 mix of 4% PFA:heptane (500 ul each if using a 1.5ml tube) for 15-20 minutes at room temperature. Keep moving using the rotating wheel (60 rpm).
6) Remove the bottom (aqueous) layer.
7) Add 800ul MeOH and shake hard for 15-20 seconds to crack the vitelline membranes. Embryos with cracked membranes will sink to the bottom. Remove the liquid (top layer first and add more MeOH and shake again if lots of embryos are still at the interface).
8) Rise 3X with MeOH
9) Store in MeOH at -20oC at least ON before use.
Note – if you will be staining with phalloiden substitute EtOH for MeOH in this procedure.
Tribolium embryo fixation
1) Collect embryos in a 300um sieve.
a. Pour onto a blank piece of paper.
b. Tap onto a second piece – leaving bits of bug and flour behind.
c. Repeat tapping until just eggs are left.
d. Transfer them to a basket.
2) Rinse well with water to remove flour on embryos.
3) Dechorionate in a 1:1 mix of household bleach and dH2O for 3 minutes.
4) Rinse well with water.
5) Fix in 1:1 mix of 4% PFA:heptane (500 ul each if using a 1.5ml tube) for 15-20 minutes at room temperature. Keep moving using the rotating wheel (60 rpm).
6) Remove the bottom (aqueous) layer.
7) Add 800ul MeOH and shake hard for 15-20 seconds to crack the vitelline membranes. Embryos with cracked membranes will sink to the bottom. Remove the liquid (top layer first and add more MeOH and shake again if lots of embryos are still at the interface).
8) Rise 3X with MeOH
9) Store in MeOH at -20oC at least ON before use.
Note – if you will be staining with phalloiden substitute EtOH for MeOH in this procedure.