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INFORMATION FOR

In Situ Hybridization - Short Fast 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

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.(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

___ 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) ___

___ Pre-hyb for 1hr 60° (trops) or 6 hrs for laevis

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

Day 2: ~4 hours to 4 hour antibody step

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

-Dip into hyb to wash off excess probe

___ 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) 4 hrs at RT. 0.2 µL/0.5 mL -SAVE antibody - 0.5 mL into a probe test tube works also!!!

Wash twice in MAB, 5 minutes, RT.
1) ___
2) ___

___ MAB wash o/n at 4oC

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

MAB wash embryos 15 minutes each RT
1) ___
2) ___
3) ___

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 RT or 4oC (500 ml per basket). Wrap in aluminum foil.

NBT/BCIP is an effective alternative and may have higher resolution

NBT – 75 mg/ml in 70% DMF
BCIP – 50 mg/ml in 100% DMF
4.5 ml/ml NBT
3.5 ml/ml BCIP

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

___ Fix 1 hr to overnight in Bouin's RT

___ Multiple 10 min washes in 1xSSC until yellow stain from Bouin's is gone. Can be done overnight. RT.

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

for 50 mls:

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

Use light box.

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

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