Whole Mount in situ Hybridization (WISH) on mouse embryos

Modified from Wilkinson and Nieto (1993) Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts, Methods in Enzymology, 225:361-373.

Day 1

1. Dissect embryos in cold PBS and fix in 4% PFA while rocking overnight.
   4% PFA
   for 500 mls
   PBS                            500 mL
   Paraformaldehyde            20 g
   10N Sodium Hydroxide    100 μL

Day2

1. Wash and dehydrate embryos through a series of 5-minute washes at room temperature:
   -two washes in PBT        
   -25% methanol (in PBT)
   -50% methanol (in PBT)
   -75% methanol (in PBT)
   -100% methanol (wash once then store in 100% methanol at -20° C)
   
   PBT
   for 500 mls
   PBS              500 ml
   Tween-20     500 μl

Day 3
 

2. Prepare dehydrated embryos:

-For 7.5 dpc, puncture the amnion

-For 9.5 dpc and older, puncture myelencephalon and telencephalon 

3. Rehydrate embryos through a series of 5-minute washes at room temperature:

-75% methanol (in PBT)

-50% methanol (in PBT)

-25% methanol (in PBT)

-two washes in PBT        

4. Bleach embryos with 6% hydrogen peroxide solution in PBT for 1 hour at room temperature while rocking.
Hydrogen Peroxide solution in PBT
for 20 mls

50% H₂O₂ solution   2.4 mls  

PBT                           17.6 mls 

4. Wash embryos with PBT 2X 5 minutes each at room temperature.

5. Treat embryos with 10 mg/ml of Proteinase K (PK) in PBT, Do Not Rock.

7.5 dpc for 1 minute in half concentration PK solution (5 μg/ml)

8.5 dpc for 3 minutes in half concentration PK solution (5 μg/ml)

9.5 dpc for 8 minutes in full concentration PK solution (10 μg/ml)

10.5 dpc for 9 minutes in full concentration PK solution (10 μg/ml)

11.5 dpc for 12 minutes in full concentration PK solution (10 μg/ml)

12.5-13.5 dpc for 15 minutes in full concentration PK solution (10 μg/ml)

6. Wash with 2 mg/ml fresh glycine in PBT for 5 minutes at room temperature, Do Not Rock.

7. Wash 2X in PBT for 5 minutes each, at room temperature, Do Not Rock.

8. Refix embryos with 0.2% glutaraldehyde/4% PFA in PBT for 20 minutes at room temperature (80 μl of 25% Glutaraldehyde in 10 mls of 4% PFA).

9. Prewarm Prehybridization/Hybridization Solution to 65° C. 
Prehybridization/Hybridization Solution

for 50 mls

RNAse free water   7.3 mls

Formamide             25 mls 

20X SSC                 12.5 mls

10% SDS                 5.0 mls

10 mg/ml tRNA      50 μl 

10 mg/ml Heparin       50 μl (10 mg/ml solution can be made, aliquoted and stored at -20° C)

Note: Can make Prehybridization/Hybridization Solution ahead of time and store at -20° C.

10. Wash embryos 2X in PBT for 5 minutes each at room temperature, resume rocking for subsequent washes.

11. Add 2 mls of prewarmed prehybridization solution and incubate for at least 1 hour at 70° C rocking/rolling in hybridization oven. 

12. Replace prehybridization solution with 1 ml fresh hybridization solution per tube and probe. Add 5 μl probe/ml hybridization solution. Incubate overnight at 70° C in hybridization oven gently rocking/rolling. 

Day 2

1. Make Solution I (made fresh each time) and Solution II (can be made ahead of time and stored at room temperature).
   Solution I
   for 20 mls
   
   Formamide             40 mls
   20X SSC                 16 mls
   10% SDS                8 mls
   H₂O                      16 mls
   
   Solution II
   for 80 mls
   
   5M NaCl                       8 mls
   1M Tris HCl pH 7.5       0.8 ml
   Tween-20                     80 μl
   Water                          71.1 mls
    
2. Prewarm Solution I to 65° C. 
3. Wash embryos 2X with Solution I for 30 minutes each at 70° C rocking.
4. Make Maleic Acid Buffer (MAB; can be made ahead of time) and MABTL (made fresh each time).
   Maleic Acid Buffer (MAB)
   for 1L
   
   Maleic Acid                          11.6 g
   5M NaCl                               30 mls
   NaOH pellets                        7.5 g
   diH₂O                                   800 mls
   -add 5M NaOH drop by drop until pH 7.5 is reached. 
   -then bring the volume to 1 L and store are room temperature. 
   Note: Autoclave buffer if contamination becomes an issue.
   
   MABTL 
   for 100 mls
   
   MAB                       100 mls
   Tween-20               100 μl
   Levamisole             50 mg
    
5. Wash with 1:1 mixture of Solution I : Solution II for 10 minutes at 70° C rocking/rolling in a hybridization oven. 
6. Wash with Solution II at room temperature for 5 minutes.
7. Wash with Solution III at room temperature for 5 minutes.
   Solution III
   for 80 ml
   
   Formamide             40 mls
   20X SSC pH 4.5       8 mls
   Water                     32 mls
    
8. Was embryos 2X with Solution III for 30 minutes at 65° C on a rocker.
9. Wash embryos 3X with MABTL for 5 minutes each at room temperature.
10. Rock embryos for 3 hours in MABTL + Block 
    MABTL + Block
    for 40 ml
    
    MABTL                                  32mls
    Roche Blocking Reagent      800mg
    -heat in microwave until starts to boil then vortex; repeat until in solution
    -add 8 mls of MABTL to bring up to 40 ml 
    -wait until cooled before using
    WARNING- solution boils very quickly so take caution not to allow boiling liquids to come in contact with skin.
     
11. Add 1 ml of anti-DIG antibody to 4 mL of block (1:4000 dilution) and incubate overnight at 4° C while rocking. 

Day 3

1. Remove antibody solution and wash in fresh MABTL 3X 5 minutes each at room temperature.
   MABTL 
   for 100 ml
   
   MAB                       100 mls
   Tween-20               100 μl
   Levamisole             50 mg
2. Wash 5-8 more times during the day with MABTL for ~1 hour each, then overnight at room temperature. 

Day 4

1. Wash 3X in NTMTL for 10 minutes each.
   NTMTL 
   for 200 mls
   
   5M NaCl                       2 mls
   1M Tris pH 9.5              10 mls
   1M MgCl2                     5 mls
   Tween-20                     100 μl
   Tetramisole                  50 mg
   Water                           82.9 mls
    
2. Replace with 1 ml BM purple AP substrate and rock gently at room temperature protected from light. 
3. Monitor signal development; can take a few hours to overnight for signal to appear.
4. When ready to stop the reaction, wash 3X with PBT for 5 minutes each.
5. Post fix in 4% PFA for minimally 1 hour at room temperature. Stained embryos can be stored in 4% PFA or washed into PBS and stored at 4° C. 

Reagents

PFA- Sigma (P6148-1KG)

Glutaraldehyde- Sigma (G6257-10ML)

Tween-20- Sigma (P1379-500ML)

Hydrogen peroxide- Sigma (516813-500ML)

Proteinase K- Sigma (03115879001)

Glycine- Sigma (50046-250G)

Tetramisole- Sigma (L9756)

Formamide- VWR (0606-950ML)

tRNA- Sigma (R-8508)

Heparin- Sigma (H6279-100KU)

anti-DIG antibody- Sigma (11093274910)

BM Purple- Sigma (11442074001)