IN-SITU HYBRIDIZATION PROTOCOL
Note: The volumes indicated in this protocol are for 1 rack of slides
DAY 1
Pre-treatment and hybridization
1. Fix slides in 4% paraformaldehyde + 0.2% glutaraldehyde* for 10 min at RT
(*Save the solution for step 7)
4% para
250 mL
Glutaraldehyde
2 mL (stock: 25 mg/100 mL)
2. Wash with PBT 3  5 min at RT
10% Tween 20
20 mL
10X PBS
200 mL
Sterile H2O
up to 2L
3. Bleach with 6% H2O2/PBT for 5 min at RT
30% H2O2
50 mL
PBT
200 mL
4. Wash with PBT 3  5 min at RT
Proteinase K
(mouse)
Age
time
E10.5  1.00
E11.5  1.30
E12.5  2.00
E13.5  2.30
E14.5  3.00
E15.5  3.30
E16.5  3.45
E17.5  4.00
P1-P5  4.30
P8-P10 
4.45
Proteinase K
(chicken)
ProK in
Age 200 mL time
E3  20 µl 2 min
E4  30 µl 2 min
E5  40 µl 2 min
E6  60 µl 2 min
E7  80 µl 2 min
E8  100 µl 2 min
E9  100 µl 2.5 ~
3 min
5. Treat with 10 µg/mL Proteinase K/PBT at RT for optimal time depending on embryonic
age
100 µL Proteinase K + 200 mL PBT (*Prot. K: Roche recombinant)
6. Quick wash with 2 mg/mL Glycine/PBT for an initial 30 sec, then wash for 10 min at RT
1 g Glycine powder in 500 mL PBT
7. Wash with PBT 3  5 min at RT
8. Fix with 4% Para + 0.2% Glutaraldehyde for 10 min at RT (omit glutaraldehyde if using
fluorescence)
9. Wash with PBT 3  5 min at RT
10. Stack slides using dividers and put them in heat sealable bags (4 slides/bag). Add 4~5 mL
of preheated (70C) prehybridization solution and 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 (Sigma Cat.# H3393-250KU)
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 into the bags and incubate overnight at 70C
DAY 2
Post-hybridization wash and Antibody binding
1. Dissemble the slides stacks and wash with prewarmed (70C) Solution 1, 3  15 min at 70C
in shaking waterbath
750 mL
1.5 L(for 2 racks)
Formamide
375 mL
750 mL
20XSSC (pH 4.5)
225 mL
450 mL
10% SDS
75 mL
150 mL
Sterile H2O
75 mL
150 mL (Prewarm @ 70C in Hyb.chamber)
2. Wash with prewarmed (65C) Solution 3, 3  15 min at 65C in shaking waterbath
750 mL
1.5 L(for 2 racks)
Formamide
375 mL
750 mL
20XSSC (pH 4.5)
90 mL
180 mL
Sterile H2O
285 mL
570 mL (Prewarm @ 65C in waterbath)
3. Wash with TBST, 3  5 min at RT
10XTBS*
100 mL
200 mL
10% tween20
100 mL
Levimasole
Sterile H2O
* 10XTBS:
NaCl
144 g
200 mL
KCl
3.6 g
0.4 g
0.8 g
1M Tris (pH 7.5)
450 mL
up to 1 L
up to 2 L
Sterile H2O
up to 2 L
4. Block with 5 % Sheep serum/TBST for 1 hr at RT
Sheep serum
10 mL
TBST
190 mL
5. Stack slides in heat sealable bags, add with 5~6 mL of 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 and Detection
1. Remove slides from bags and wash wit TBST, 3  5 min and 3  30 min at RT
10XTBS*
200 mL
10% tween20
* 10XTBS:
NaCl
144 g
200 mL
KCl
3.6 g
Levimasole
0.8 g
1M Tris (pH 7.5)
450 mL
Sterile H2O
up to 2 L
Sterile H2O
up to 2 L
2. Wash with NTMT, 3  10 min at RT
5M NaCl
30 mL
40 mL
2M Tris HCl (pH 9.5)*
75 mL
100 mL
1M MgCl2
15 mL
20 mL
10% Tween20
15 mL
20 mL
Levimasole
0.72 g
0.96 g
Sterile H2O
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
3. Incubate* (on a shaker) with NBT+BCIP/NTMT overnight 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 container box with foil.
DAY 4
1. Stop reaction with 1 mM EDTA/PBT for 30 min at RT
0.5M EDTA
500 µL
PBT
250 mL
2. Mount dried slides with coverglass (2460mm, No. 0) using gelvitol and let dry overnight