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INFORMATION FOR

In Situ Hybridization - Short Protocol

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Fixation of embryos - long term storage:

Fix embryos in MEMFA for 1-2 hr:

1xMEMFA:

1/10 vol 10xMEMFA salt
1/10 vol formaldehyde
8/10 vol water

Dehydrate in MeOH (Change MeOH several times)
Embryos can now be stored long term at –20°C

BEGIN IN SITU

DAY 1: ~3 hours needed until 6 hr. prehyb step

Reagents:

Thaw paraformaldehyde 20% (10-20 ml)
Set water bath to 60oC (shaker)
Make 1 Liter 1xPBS+0.1% Tween-20 (PTw)
Thaw Proteinase K 10mg/ml (50-100 µl)
Methanol
0.1M Triethanolamine pH 7-8 (~100-200 ml)
Acetic Anhydride (125-250 µl)
Hyb Buffer (50-100 ml)

Rehydration: (PTw=1xPBS+0.1%Tween-20)

5 minutes each wash each RT (room temperature)
___ Methanol (MeOH)
___75% MeOH 25% H2O
___50% MeOH 50% H2O
___25% MeOH 75% PTw
___100% PTw

3x 5 minutes in PTw RT
1) ___
2) ___
3) ___

___ Proteinase K ~5 min. RT
(100µl 10mg/ml PK/100ml PTw)

Rinse 2x 5 minutes in 0.1M triethanolamine pH 7-8 RT
1) ___
2) ___

___ Add 12.5µl acetic anhydride/5mls: 5 minutes (125µl in 50 ml/250µl in 100 ml) - in triethanolamine RT

c Repeat acetic anhydride addition: 5 minutes RT
Wash 2x 5 minutes in PTw RT
1) ___
2) ___

___ Refix for 20 minutes with 4% paraformaldehyde in PTw (10 ml 20% PF into 40 ml PTw) RT

Wash 5x 5 minutes in PTw (wash off excess paraformaldehyde) RT
1) ___
2) ___
3) ___
4) ___
5) ___

___ Prehybridize 6 hours 60°C. (save prehyb solution on your bench) (can shorten to 1 hr for trops)

___ Replace with 0.5ml probe solution (1µg/ml probe), hybridize overnight at 60°C.

Day 2: ~4 hours

Reagents:

2xSSC (100 ml 20xSSC into 900 ml H2O)
Thaw RNAse A (20 mg/ml)/RNAse T1 (10 mg/ml) (Both at 1000x)
Thaw MAB + 10% BMB Blocking agent (5-10 ml)
2xSSC with RNase A 20 µg/ml, RNAse T1 10 µg/ml
0.2xSSC (100 ml 2xSSC into 900 ml H2O)
MAB pH 7.5

___ Remove probe and keep for reuse
___ Replace probe with hyb. buffer), Incubate 60°C for 3 minutes
___ Wash 3 minutes 2xSSC, 60°C
___ Wash 3 minutes 2xSSC, 60°C

Washes: 3x 20 minutes 2xSSC, 60°C
1) ___
2) ___
3) ___

___ Wash 30 minutes (37oC) 2x SSC with RNase A 20 µg/ml, RNAse T1 10 µg/ml

___ Wash once in 2x SSC 10 minutes room temp.
Wash twice in 0.2x SSC 30 minutes, 60°C.
1) ___
2) ___

Wash twice in MAB, 10-15 minutes, RT.
1) ___
2) ___

___ Wash in MAB + 2% BMB Blocking Reagent (10 ml 10% BMB/1xMAB to 50 ml with MAB) 1 hr or more, RT.

___ Replace with MAB + 2% BMB Blocking Reagent + Ab (1/3000 dilution of the anti-digoxygenin AP antibody) overnight at 4oC (or 4 hrs at RT.) 0.2 µL/0.5 mL -SAVE antibody - 0.5 mL into a probe test tube works also!!!

DAY 2.5-3 (5.5 hours)

Reagents:

Thaw Alkaline Phosphatase buffer (with 0.005 M levamisol and 0.1% Tween) (100-200 ml)
MAB - Maleic Acid Buffer pH 7.5

Wash embryos at least 5 times, 1 hour each with MAB RT
1) ___
2) ___
3) ___
4) ___
5) ___

Wash twice, 5 minutes each at room temperature with Alkaline phosphatase buffer (make sure levamisol 0.005 M and 0.1% Tween have been added) RT
1) ___
2) ___

___Replace last wash with BM purple

Stopping Color Reaction:

Reagents:

MAB (malaeic acid buffer)
Bouin's Fixative (70 ml saturated Picric acid/5 ml glacial acetic acid/25 ml formaldehyde)
1xSSC (1 liter)
EtOH
Bleach Solution (1% H2O2, 5% formamide, 0.5x SSC)
20xSSC
formamide
30% peroxide

___ Stop chromogenic reaction with quick wash in MAB RT
___ Fix overnight in Bouin's RT
___ Multiple 10 minute washes in 70% buffered EtOH RT

Rehydrate stepwise into 1X SSC: RT

___ 5 min 75% buffered EtOH: 25% 1X SSC
___ 5 min 50%:50%
___ 5 min 25%: 75%
___ 5 min 100% 1xSSC
___ 5 min once more in 1xSSC

___ Bleach embryos in bleaching soln. (~1-2 hours):

for 50 mls:

1.25 ml 20xSSC
2.5 ml formamide
2ml 30% peroxide
45 ml distilled water

Use light box. RT

Wash twice for 5 min in 1xSSC RT
1) ___
2) ___

___Replace last wash with BM purple

___Take pictures of uncleared or clear embryos in BB:BA clearing agent (benzyl benzoate/benzyl alcohol).