Prior to in situ:

  • Fix embryos in freshly made PFA or“thawed” PFA 24 hrs after collecting embryos at RT in glass vials until ready to be wash with MetOH.
  • Transfer embryosaccordingly to each solns below
  1. 1X 10 mins30%Methanol/DEPC
  2. 1X 10 mins50%Methanol/DEPC
  3. 1X 10 mins70%Methanol/DEPC
  4. 1X 10 mins100% Methanol
  5. 100% Methanol (embryos can be store here in -20˚C for further use)

IN SITU:

Day 1:

  1. Transfer fixed embryos from glass vials into RNase free micro-centrifuge tubes (in RNase free cabinet); turn on water bath and set it to 70˚C.
  2. Wash embryos at room temperature using 1ml on shaker:
  3. 1X 5 mins75%methanol/PBT
  4. 1X 5 mins 50%methanol/PBT
  5. 1X 5 mins 25%methanol/PBT
  6. 3X 5 mins each100%PBT
  7. Thaw out 4% PFA in water bath; take out when thawed.
  8. Digest with Proteinase-K according to the stage of development at room temp.
    12 somites to 24h10 minutes
    30-36hs15 minutes
    48+20minutes
  9. Add 0.5ul of 20mg/ml of stock proteinase-K into 1 ml 1X PBT (want final concentration to be 10ug/ul)
  10. REMEMBER: add 1ml 1X PBT into vials first before adding 0.5 ul of 20mg/ml of stock proteinase-K
  11. Aspirate (take out) proteinase-K immediately and refix with 4% PFA for 20 minutes at room temperature.
  12. Prewarm pre-hybridization and hybridization+tRNA at 70˚C in water bath.
  13. Rinse 5X in 1X PBT for five minutes each at room temperature.
  14. Rinse in pre-hybirdization one time for five minutes at 70˚C in water bath.
  15. Aspirate and transfer new pre-hybridization to micro-centrifuge tubes and incubate in water bath for three hours at 70˚C.
  16. Twenty minutes prior to finishing 3 hrs incubation, prepared probe in hybridization+tRNA and incubate (heat shock) at 80˚C for ten minutes
  17. Aspirate pre-hybridization solution and replace with probe+hybrdization+tRNA. Incubate at 70˚Cin water bath for at least 16 hrs.

Day 2:

  1. Turn on Hybridization oven to 70˚C and do the following washes in Hyb. oven
  2. 10min 75% hyb mix+25% 2X SSC
  3. 10 min 50% hyb mix+50% 2X SSC
  4. 10 min 25% hyb mix+75% 2X SSC
  5. 10 min 100% 2X SSC
  6. Wash 2X for 15 minutes each with 0.2X SSC at 70˚Cin hybridization oven (dilute 2X to needed soln w/ DEPC-treated water)
  7. Thaw out PI Buffer if needed or make PI Buffer
  8. Wash 2X for 5 minutes each with 50% 0.2X SSC+50% PBT at room temp
  9. Wash 2X for 5 minutes each with 100% PBT at room temp
  10. Incubate embryos in Pre-Incubation (PI) buffer for 5 min at room temp. (PI buffer= for 50 ml 49ml of PBT add 1ml sheep serum and 100mg Bovine SAlbumin in fridge stored at -20˚C)
  11. Aspirate and replace with new PI buffer and incubate for 1 hour at room temp.
  12. Incubate with a 1:5000 final dilution in PI PRE-ABSORBED anti-DIG antibody at 4˚C overnight on the rocker. (Dilute stock of antibody (1:100)  for 1.0ml, add 20ul of anti-bodyinto 980ul PI buffer)
  13. Remember: Add PI buffer first before adding antibody.

Day 3:

  1. Wash 8X for 15 minutes each wash with PBT @ RT
  2. Wash 3X for 5 minutes DIG AP Buffer (ALWAYS made fresh) at room temperature.Use “DIG soln” box. Stock is 10X  dilute to 1X DIG AP Buffer
  3. To make 40 ml  4 ml 10X DIG + 36 ml SDW
  4. After the third wash with DIG AP buffer, make AP substrate (NBT/BCIP tablet) solution at room temp and cover it in foil. Keep it in the dark on rotor.
  5. After last wash of DIG AP buffer, transfer samples to wells in micro-plate with big plastic pipette.
  6. Remove all DIG AP buffer making sure to not expose embryos to air for more then a few seconds
  7. HINT: do one well at a time.
  8. Add AP substrate (600 ul/well) to wells and cover microplate wells with aluminum foil. Set wells back onto shaker.
  9. HINT: Use new glass pipette during each new experiment
  10. Monitor color development every thirty minutes
  11. Stop reaction by washing with PBT quickly three times
  12. Fix embryos in 4% PFA overninght.