WHOLE-MOUNT IN-SITU HYBRIDIZATION
DAY 1: Pre-treatment and hybridization
1. Using forceps or a needle, make a hole in the roof of the hindbrain and open the forebrain
2. Rehydrate embryos by rocking 5 min in 75%, 50%, 25% MetOH/PBT, then PBT 2 x 5 min
10% Tween 20
20 mL
10X PBS
200 mL
Sterile H2O
up to 2L
3. Bleach with 6% H2O2/PBT for 1 hr at RT, rocking (25 mL 30% H2O2 in 100 mL PBT)
(make 4% paraformaldehyde for step 8)
4. Wash with PBT 3  5 min at RT
5. Treat with an optimal concentration of Proteinase K/PBT at RT for 15 min
Mouse (1X = 100µL Prot. K/100mL PBT)
Chicken (1X = 50µL Prot. K/100mL PBT)
pre-turn = 0.1X
Turning = 0.5X
Otic cup = 1.0X
Otocyst = 1.5X
11~15 somites = 1/4X 20~30 somites = 1/2X
E3~E3.5 = 1.0X
E4m = 1.5X
E4.5 = 2.0X
E5 = 2.5X
E6 = 3.0X
E7 = 4.0X
6. Quick wash with 2 mg/mL Glycine/PBT for an initial 30 sec, then wash 2 x 5 min at RT
(thaw glutaaldehyde)
1 g Glycine powder in 500 mL PBT
7. Wash with PBT 2  5 min at RT
8. Fix with 4% Paraformaldehyde + 0.2% Glutaraldehyde for 10 min at RT (omit
glutaraldehyde if using fluorescence) (2 mL 25% Glutaraldehyde in 250 mL paraformaldehyde)
9. Wash with PBT 2  5 min at RT
10. Transfer embryos to 1.5 mL o-ring screw-topped microfuge tubes
11. Add 1 mL of preheated (70C) Prehybridization solution and mix gently by inversion.
Remove and add another 1 mL. Incubate in hybridization chamber for 1 hr at 70C
(final conc.)
Formamide
25 mL
50%
20XSSC (pH 4.5)*
15 mL
5X (*1.5 L 20XSSC pH7 + 300 mL 1M citric acid)
Heparin (20 mg/mL)
125 µL
50 µg/mL
Yeast RNA (5 mg/mL)
0.5 mL
50 µg/mL
10% SDS
5 mL
1%
DEPC H2O
up to 50 mL
10. Add RNA probes (1 µg/ 1 mL) into the tubes and incubate overnight at 70C
DAY 2: Post-hybridization wash and Antibody binding
1. Rinse original scintillation vials with Solution 1. Transfer embryos from the tubes into prewarmed solution1 in scintillation vials. Wash with prewarmed (70C) Solution 1, 2  30
min at 70C in shaking waterbath
500 mL
750 mL
1L
1.5 L
Formamide
250 mL
375 mL
500 mL
750 mL
20XSSC (pH 4.5)
150 mL
225 mL
300 mL
450 mL
10% SDS
50 mL
75 mL
100 mL
150 mL
Sterile H2O
50 mL
75 mL
100 mL
150 mL
2. Wash with prewarmed (65C) Solution 3, 2  30 min at 65C in shaking waterbath
500 mL
750 mL
1L
1.5 L
Formamide
250 mL
375 mL
500 mL
750 mL
20XSSC (pH 4.5)
60 mL
90 mL
120 mL
180 mL
Sterile H2O
190 mL
285 mL
380 mL
570 mL
3. Transfer embryos to 6 well plates with mesh. Wash with TBST, 3  5 min at RT, rocking
10XTBS*
100 mL
10% tween20
* 10XTBS:
NaCl
160 g
100 mL
KCl
4g
Levamisole
0.4 g
1M Tris (pH 7.5)
500 mL
Sterile H2O
up to 1 L
Sterile H2O
up to 2 L
4. Block with 10 % Sheep serum/TBST for 2.5 hrs at RT
Sheep serum 10 mL
TBST
90 mL
5. Add anti-Digoxygenin antibody, and incubate overnight at 4C
a. Weigh out 60 mg Mouse (or Chicken) embryo extract in 15 mL tube
b. Add 10 mL TBST
c. Rock (incubate) at 70C for 30 min and then transfer to ice bucket
d. Add 100 µL of HISS (heat inactivated sheep serum)
e. Add 20 µL of anti-Dig Ab
f. Rock (incubate) for 1 hr in cold room
g. Spin 15 min, 4000 rpm, 4C using swing bucket rotor
h. Transfer supernatant into 50 mL tube
i. Add 30 mL TBST (or up to 40 mL)
DAY 3: Post-Antibody washes
1. Wash embryos with TBST 3  5 min
10XTBS*
200 mL
10% tween20
* 10XTBS:
NaCl
160 g
200 mL
KCl
4g
Levamisole
0.8 g
1M Tris (pH 7.5)
500 mL
Sterile H2O
up to 2 L
Sterile H2O
up to 2 L
2. Wash embryos with TBST 5 x 1~1.5 hrs RT
3. Wash embryos with TBST overnight at 4C
DAY 4: Detection
1. Wash with NTMT, 3  10 min at RT
5M NaCl
10 mL
20 mL
30 mL
40 mL
2M Tris HCl (pH 9.5)*
25 mL
50 mL
75 mL
100 mL
1M MgCl2
5 mL
10 mL
15 mL
20 mL
10% Tween20
5 mL
10 mL
15 mL
20 mL
Levamisole
0.24 g
0.48 g
0.72 g
0.96 g
Sterile H2O
up to 0.5 L
up to 1 L
up to 1.5 L
up to 2 L
* 2M Tris HCl (pH 9.5): 484.56g Tris + 64 mL 6N HCl + Sterile H2O up to 2L
2. Incubate* (on a shaker) with NBT+BCIP/NTMT at RT
a. Make 70% DMF (dimethyl formamide)  700 (or 1050) µL DMF + 300 (or 450) µL H2O
b. Make NBT/DMF  0.075 (or 0.113) g NBT in 1 (or 1.5) mL 70% DMF
c. Add 675 µL NBT/DMF and 525 µL BCIP in 200 mL NTMT
* Note: NBT and BCIP are light-sensitive. Wrap 6 well plates with foil.
3. Monitor after 15 min, 30 min, 1 hr, 2 hrs so on.
4. Stop reaction with 1 mM EDTA/PBT for 2 x 5 min at RT
0.5M EDTA
500 µL
PBT
250 mL
5. Wash in 50% and 80% Glycerol/PBT (0.02% NaN3) and store at 4 C