​Jadera haematoloma embryo fixation and in situ hybridization

1          Embryo fixation 

1. Collect your aged embryos from the cotton and transfer about 100-200 uL of embryos to each tube.
2. Set about 600 mL of water to boil on the hot plate.
3. Add ~1200 uL water to each tube of embryos. Tap tube to submerge embryos.
4. Submerge tubes of embryos in boiling water bath for 3 minutes.
5. 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.
6. Remove all water from tubes and replace with PBST.
7. 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.

1. Rehydrate embryos from MeOH to PBST (remove 500 uL MeOH, add 500 uL PBST, let wash 5 min. Repeat 2-3 times)
2. Transfer embryos in PBST to dissection plate, and manually remove eggshells under the scope using very fine forceps.
3. 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

1. Warm up some hybridization buffer (1 mL per tube of embryos) in the 65°C hyb oven
2. 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)
3. Rinse 3X in PBST
4. Remove PBST and add 1 mL 4% PFA, let wash 1.5 h at RT
5. Remove PFA, rinse 3X with PBST
6. Remove PBST, add 1 mL pre-warmed hyb buffer
7. Let embryos rock for at least 5 min, or until they sink in the hyb buffer
8. Let embryos incubate in 65°C incubator for at least 30 min, not longer than a few hours.
9. 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                      [  ][  ][  ]


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