Whole mount in situ short cut procedure

First day RNase free conditon

1. Pre-treatment
   100mls each in a DEPC treated containers
   
   1. 100% MetOH 5 min
      
   2. 75% MetOH2 / 25%DEPC-dH2O 5 min with agitation on a mild mixer
      
   3. 50% MetOH2 50%DEPC-dH2O 5 min with agitation on a mild mixer
      
   4. 25% MetOH2 75%PTw 5 min with agitation on a mild mixer
      
   5. PTw 5 min X 3
      
2. ProK treatment
   
   1. 10礸/ml Proteinase K in PTw ( 0.5ml in a polypropylene tube) for 5~10minutes at room temp.
      
   2. Just rinse with PTw in a rack
      
   3. 0.1M TEA 5min
      
   4. 0.1M TEA 5min
      
   5. Add 250祃 of acetic anhydride 5min
      
   6. Add 250祃 of acetic anhydride 5min
      
   7. PTw 5min with agitation X 2
      
   8. 4% formaldehyde in PTw 20min with agitation
      
   9. PTw 5min with agitation X 5
      
3. Hybridization
   
   1. PTw 500兪l+H.S 125兪l agitate 5min
      
   2. 500兪l H.S. 10min at 60亷.
      
   3. 500兪l H.S. 6 hours at 60亷.
      
   4. Probe (0.3~0.5兪g) in 500兪l H.S. overnight (12~16 hours) at 60亷
      

Second day

丂

1. 500兪l H.S. 10 min at 60亷 with aggitation in hybridization oven.
   
2. 2亊SSC丂20min at 60亷丂亊3
   
3. 0.2X SSC and agitate for 30 minutes at 60亷
   
4. MAB for 15minutes at room temperature.
   
5. MAB for 15minutes at room temperature.
   
6. O.5ml of MAB-B (MAB containing 2% BM Blocking reagent) in glass vials .
   丂
7. 0.5mls MAB-B /20% heat-inactivated sheep serum for 1hour at room temperature.
   
8. 0.5ml MAB-B/20% heat-inactivated sheep serum with anit-DIG antibody (1/2000) overnight with agitation at 4亷 (in a cold room).

Third day

Antibody wash

1. Rinse embryos with MAB.
   
2. Fill the vials with MAB 1hr RT. X5
   

Coloring reaction with BM purple

丂
1. 0.5 ml BM purple,for 5min cover with alminum foil
   
2. 0.5 ml of new BM purple.
   
3. Cover the vial with alminum foil to cut lights
   
4. 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.

丂