In situ hybridization

From dehydrated E9.5 embryos, rehydrate embryos in 75%, 50%, 25% MeOH/PBT 5min each shaking
Wash 2x PBT 5min RT
Bleach with 6% H2O2 in PBT 1h RT (~6ml each vial)
Wash 3x PBT 5min RT
Permeabilize with 10ug/ml Proteinase K in PBT RT (2.5min for E7.5, 5min for E8, 10min for E9, 20min for E10-11)
Wash with 2mg/ml Glycine in PBT 15min RT to stop ProK reaction
Wash 2x PBT 5min
Postfix with 4% PFA/ 0.2% gluteraldehyde in PBT 20min RT
Wash 2x PBT 5min
Pre-hybridize with warm hybridization buffer 1h
Add 2ml probe to each vial
Hybridize O/N 70C

Remove probes from vials and keep at -20C to reuse 5-6 times
Rinse with warm Wash Solution I
Wash with Wash Solution I 3x 30min 70C shaking
Wash with Wash Solution II 3x 30min 65C
Wash with TBST 3x 5min RT
Preblock with 10% heat-inactivated sheep serum in TBST 1-2.5h
Remove block. Add anti-dig antibody 1:5000 in 1% sheep serum
Rock O/N 4C

Wash embryos in TBST 3x 5min RT
Wash 6-8x 30min-1h TBST RT
Wash O/N TBST 4C

Wash with fresh NTMT 3x 10min
Incubate with RT BM Purple. Cover with foil. RT-37C
Check after 20min
Develop till desired darkness
Rinse briefly in NTMT 2x 5min
Wash with PBT pH5.5 2x 10min RT to stop reaction
Post fix with 4% PFA 1h RT or 4C O/N
Wash 2x PBT
Dehydrate through Methanol row. Keep at -20C

DEPC water
0.1% DEPC in H2O. Sit O/N. Autoclave

PBT
PBS (DEPC) with 0.1% Tween 20. Filter

Hybridization solution (500ml)
Formamide 50% 250ml
20x SSC pH4.5 5x 125ml
50mg/ml yeast tRNA 50ug/ml 500ul
10% SDS 1% 50ml
50mg/ml heparin 50ug/ml 500ul
DEPC H2O 74ml
Filter
Aliquot into 50ml and store at -20C

Wash solution I
Formamide 50%
20X SSC pH 4.5 5x
10% SDS 1%
DEPC H2O

Wash solution 2
Formamide 50%
20X SSC pH4.5 2x
Tween 20 0.1%
DEPC H2O

NTMT (100ml)
5M NaCl 100mM 2ml
1M Tris pH9.5 100mM 10ml
1M MgCl2 50mM 5ml
Tween 20 0.1% 0.1ml
1M Levimasole 2mM 200ul
H2O (non-DEPC)
Filter