Callosobruchus maculatus Rearing, Embryos Collection, Fixation, Dissection
            Jie Xiang, Iain Forrest, Ebony Argaez
​
Rearing of C. mac
  1. C. mac were bought from Carolina Biological Supply Company
  2. Cover the bottom of the petri dish with a single layer of organic mung beans* and introduce at least 15 females and 15 males
*Organic mung beans from whole foods
  1. Keep the cultures in a  250C or 300C incubator
  2. Replace mung beans around every two to three weeks
    1. From the petri dish, collect larva/pupa and save
    2. discard about half of the beans
    3. replace discarded beans until bottom of petri dish is covered with a single layer of beans
  3. Disposal of cultures
    1. Place in -200C for ~72 hours prior to disposal
Embryo Collection
  1. To set up a collection, place 36 males and 27 females from the colony in a medium sized petri dish (100mm x 15mm)
  2. Place 9 hosts, orange beads into the petri dish and leave the petri dish in a 250C 300C incubator. After the appropriate time window, the beads will be ready for embryo collection.
  3. Fold a piece of filter paper (100 mm x 15mm) in half, twice and then unfold to create a crease, place this into a new petri dish (100 mm x 15 mm)
  4. To collect the embryos, hold an orange bead with a forcep inside the hole and brush off eggs onto the filter paper. A brush with a blunt end works best.
  5. Collect any leftover eggs from the petri dish and brush onto filter paper.
  6. For RNA extraction:
    1. Transfer embryos to an RNase free tube (1.5ml blue tube for homogenizing)
    2. Add 1ml of RNAlater
    3. Leave at 40C overnight*
*embryos can be saved in RNAlater for up to one month at 40C and for long term storage, the tube should be left in -800C or -200C freezer after 40C overnight
  1. For Fixation: Place in 1.5 ml Eppendorf tube
Embryo Fixation
  1. To the 1.5ml Eppendorf tube, Add 50% bleach for 1 min. Tilt tube occasionally *
  2. Rinse with tap water two times*
*Make sure to use small pipette tip to remove bleach and water in order to not lose embryos
  1. Add 1 ml of dH 2 O (Note: use ~ 200 µl of embryos in each tube. If there are too many embryos, split them to more tubes)
  2. Submerge the tube with embryos in boiling water for 3 minutes.
  3. Transfer the tube to ice immediately and let it stay on ice for 7 minutes.
  4. Remove dH2O without losing embryos.
  5. Add 500 µl of heptane and 500 µl of 4% PFA (paraformaldehyde).
  6. Fix the embryos on the shaker at high speed (250 rpm) for 20 minutes.
  7. Remove the aqueous phase (PFA, lower layer) without sucking up embryos.
  8. Add 800 µl of 100% MeOH. Cap the tube and shake it vigorously for 20 seconds.
  9. 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.
  10. Remove as much solution as possible without losing any embryos.
  11. Rinse twice with 800 µl of 100% MeOH. Make sure to rinse embryos off the wall of the vial.
  12. Add 800 µl of 100% MeOH and store the tube at -20° C.
Embryo Dissection
Embryos will be dissected in PBST to remove the eggshell before staining, or in situ hybridization
  1. Remove 1.5 ml Eppendorf tube from freezer. Each tube should contain ~ 200 µl embryos.
  2. Remove MeOH, and rinse once with 500 µl of MeOH.
  3. Remove, rinse once with 500 µl of MeOH/PBST 1:1.
  4. Remove, rinse three times with 500 µl of PBST.
  5. Transfer embryos to a multi-well glass plate using a P1000 pipette tip with the end cut off.
  6. To remove egg shell, hand-dissect embryos in PBST using Dumont #5 forceps.
  7. Transfer embryos back to a 1.5 ml Eppendorf tube.