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

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

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

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
Prewarm Solution I to 65° C.
Wash embryos 2X with Solution I for 30 minutes each at 70° C rocking.
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
Wash with 1:1 mixture of Solution I : Solution II for 10 minutes at 70° C rocking/rolling in a hybridization oven.
Wash with Solution II at room temperature for 5 minutes.
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
Was embryos 2X with Solution III for 30 minutes at 65° C on a rocker.
Wash embryos 3X with MABTL for 5 minutes each at room temperature.
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.
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

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
Wash 5-8 more times during the day with MABTL for ~1 hour each, then overnight at room temperature.

Day 4

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
Replace with 1 ml BM purple AP substrate and rock gently at room temperature protected from light.
Monitor signal development; can take a few hours to overnight for signal to appear.
When ready to stop the reaction, wash 3X with PBT for 5 minutes each.
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)