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- Fix embryos with 4% formaldehyde in 0.25XPBS for 60 to 90 minutes (max) at room temperature. For embryos at mid stage 9 or older, incubate them in relaxant medium for 5 minutes at 4o C prior to fixing them with cold fixative (same fixative as above).
- Rinse with PBT (1X PBS, 1% triton 100).
- Devitellinize embryos in PBT . (Can save the embryos in PBT for a week)
- Treat embryos for 40 minutes with 50ul of protein kinase (1ul PK 20mg/ml in 1600ul 1XPBS) at room temperature.
- Pre-incubate embryos with 100ul of Antibody serum* (1XPBS, 10% NGS, 1% triton X-100) for several hours (3-6hrs) at room temperature or O/N at 4o C
- Replace with ~250-500ul of primary mouse anti-histone antibody (1ul of anti-histone antibody in 1ml antibody serum). Incubate overnight with constant rolling, at room temperature.
- Rinse every hour with 100ul antibody serum for 4 to 8 hours.
- Replace with secondary HRP conjugated rabbit anti-mouse antibody (1ul of secondary antibody in 500ul antibody serum). Incubate O/N at room temperature with constant rotating, same as for primary antibody).
- Rinse once every hour for 2 hours at room temperature.
- Do HRP reaction immediately after rinsing.
Horse Radish Peroxidase (HRP) staining
- Wear gloves
- Get an aliquot of DAB in the -20 freezer.
- Each aliquot contains 250ul of 2mg/ml DAB. Centrifuge the aliquot so that the DAB solution is located at the bottom of the tube. Add 750ul of PBS.
- Make a solution of 1%-3% H2O2 in an eppendorf tube.
- Prepare waste container with bleach in it.
- Take a 12 wells porcelain dish and put 100ul of DAB in each well
- Transfer embryos to the wells, incubate 10-20 minutes in DAB. Cover wells with aluminum foil when incubating.
- Add 3ul of 1% H2O2 in each well and monitor the embryos in each well for staining.
- When desired staining coloration is reached, pipette out the DAB (make sure to leave the embryos in the well) and discard DAB into the waste bottle containing bleach.
- Rinse the embryos with PBS.
- Transfer embryos in eppendorf tube with fresh PBS.
- Embryos are ready for dehydration or for transfer in glycerol.