Jadera haematoloma embryo fixation and in situ hybridization
1 Embryo fixation
1 Embryo fixation
- Collect your aged embryos from the cotton and transfer about 100-200 uL of embryos to each tube.
- Set about 600 mL of water to boil on the hot plate.
- Add ~1200 uL water to each tube of embryos. Tap tube to submerge embryos.
- Submerge tubes of embryos in boiling water bath for 3 minutes.
- Carefully (but quickly) move tubes to ice and submerge. As you are transferring to ice, you can remove most of the tap water using a glass pipette or add ace to the tubes - this will help them cool down faster. Leave for ~ 7 min or until cold.
- Remove all water from tubes and replace with PBST.
- Under a dissecting microscope, carefully remove a piece of each chorion. See video below for example.
8. Remove PBST, add 4% paraformaldehyde (PFA); rock for 30 min
9. Rinse several times with PBST
10. Transfer to methanol: Remove 500 uL PBST, add 500 uL MeOH, let wash 5 min. Repeat 2-3 times, then rinse in methanol. Store at -20˚C in methanol.
2 Chorion removal
We usually spend a day in between fixation and in situ removing embryos from their eggshells manually.
- Rehydrate embryos from MeOH to PBST (remove 500 uL MeOH, add 500 uL PBST, let wash 5 min. Repeat 2-3 times)
- Transfer embryos in PBST to dissection plate, and manually remove eggshells under the scope using very fine forceps.
- Move embryos back to tube and dehydrate into MeOH (remove 500 uL PBST, add 500 uL MeOH, let wash 3 min. Rinse a couple times in MeOH and store in MeOH). I like to mark the tube in some way that lets me know these embryos are free of eggshells.
3 In situ hybridization
Day 1
- Warm up some hybridization buffer (1 mL per tube of embryos) in the 65°C hyb oven
- Rehydrate your embryos from 100% MeOH (remove 500 uL MeOH, add 500 uL PBST, let wash 5 min. Repeat this 2-3 times until solution is ~100% PBST)
- Rinse 3X in PBST
- Remove PBST and add 1 mL 4% PFA, let wash 1.5 h at RT
- Remove PFA, rinse 3X with PBST
- Remove PBST, add 1 mL pre-warmed hyb buffer
- Let embryos rock for at least 5 min, or until they sink in the hyb buffer
- Let embryos incubate in 65°C incubator for at least 30 min, not longer than a few hours.
- Remove hybridization buffer and add at least 200 uL of probe (typically diluting the original 20 uL of stock probe solution 1:5000 works well) and place vertically in the 65°C incubator overnight.
Day 2
Put 2 wash volumes hyb buffer and 1 wash volume 2X SSC in the hybridization oven to warm up.
Wash volume = 1 mL/tube of embryos. Remove probe and perform the following washes:
Buffer Temperature (C) Time
Hyb buffer (pre-warmed) 65 ≥20 min [ ]
Hyb buffer (pre-warmed) 65 ≥20 min [ ]
2X SSC (pre-warmed) 65 ≥20 min [ ]
2X SSC RT ≥20 min [ ]
0.2X SSC RT ≥20 min [ ]
PBST RT 3X quick rinse [ ][ ][ ]
10% sheep serum in PBST RT ≥30 min [ ]
1:1600 anti-dig in 10% sheep serum RT ≥1 h [ ]
PBST RT 3X quick rinse [ ][ ][ ]
PBST 4 overnight* [ ]
*If you want to keep going, at this point you can proceed to the Day 3 washes instead of putting embryos at 4 C overnight.
Day 3
Make your AP staining buffer fresh, then perform the following washes:
Buffer Temperature (C) Time
PBST RT ≥10 min [ ]
PBST RT ≥10 min [ ]
PBST RT ≥10 min [ ]
PBST RT ≥10 min [ ]
PBST RT ≥10 min [ ]
AP staining buffer RT ≥5 min [ ]
AP staining buffer RT ≥5 min [ ]
AP staining buffer RT ≥5 min [ ]
Remove all buffer and add 500 uL of freshly made NBT/BCIP staining solution. [ ]
Time for stain development needs to be determined empirically, but it usually takes several hours. I will often perform Day 2 and Day 3 washes in one day, then leave embryos to stain overnight at room temperature. It is ok if the embryos over-stain a little because a lot of background stain will be removed by the alcohol washes.
When embryos have stained enough remove staining solution to hazardous waste [ ]
Rinse 3X in PBST (rinsate should go in hazardous waste container as well). [ ][ ][ ]
Perform the following alcohol washes:
Solution Temperature (C) Time
1:1 PBST:MeOH RT ≥5 min [ ]
2X rinse in MeOH RT rinse [ ][ ]
EtOH RT rinse [ ]
2X rinse in MeOH RT rinse [ ][ ]
1:1 PBST:MeOH RT ≥5 min [ ]
Rinse 3X in PBST RT rinse [ ][ ][ ]