Whole mount in situ short cut procedure
First day RNase free conditon
Second day
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- 500ƒÊl H.S. 10 min at 60Ž with aggitation in hybridization oven.
- 2~SSC@20min at 60Ž@~3
- 0.2X SSC and agitate for 30 minutes at 60Ž
- MAB for 15minutes at room temperature.
- MAB for 15minutes at room temperature.
- O.5ml of MAB-B (MAB containing 2% BM Blocking reagent) in glass vials .
@- 0.5mls MAB-B /20% heat-inactivated sheep serum for 1hour at room temperature.
- 0.5ml MAB-B/20% heat-inactivated sheep serum with anit-DIG antibody (1/2000) overnight with agitation at 4Ž (in a cold room).
Third day
Antibody wash
- Rinse embryos with MAB.
- Fill the vials with MAB 1hr RT. X5
Coloring reaction with BM purple
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- 0.5 ml BM purple,for 5min cover with alminum foil
- 0.5 ml of new BM purple.
- Cover the vial with alminum foil to cut lights
- Check the embryos for staining repeatedly and stop the reaction when the staining condition is apropriate with 1ml of MEMFA or 4% formaldehyde in PBS.
Store in Methanol.
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