Albertson Lab In Situ Hybridization Protocol
Day 0
Prepare Embryos:
Fix embryos in 4% paraformaldehyde (PFA) o/n
Wash 3 x 10mins in PBSt
Dehydrate embryos
 1hr in 25% Methanol/ 75% PBSt
 1hr in 50% Methanol/ 50% PBSt
 2 x 1hr 100% Methanol
Store in 100% Methanol at -20°C (at least overnight)
Day 1
Rehydrate embryos:
 2 x 10mins in 50% Methanol/ 50% PBSt
 1 x 10mins in 25% Methanol/ 75% PBSt
 2 x 10mins PBSt
Dechorionate embryos if needed (Younger Embryos):
Bleach (Older Cichlid Embryos):
 10% Bleach solution of Peroxide in PBSt
 Bleach with the top open and until the dark pigments brown out.
 Rinse twice with PBSt
Proteinase K Digestion:
 Use a 1:5000 dilution of a 50mg/mL stock in PBSt
Table of Digestion Times
Zebra Fish
Though tail bud
1-14 somites
15-22 somites
24hrs
36hrs
2days
3days
4days
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30 sec
1-3mins
4-7mins
~10mins
~13mins
~16mins
~22mins
~25mins
2dpf
3dpf
4dpf
5dpf
6dpf
7dpf
Rinse twice in PBSt
Refix in 4% PFA for 30 mins
Wash 3 x 5mins in PBSt while rocking
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Cichlids
~5mins
~15mins
~17mins
~23mins
~26mins
~29mins
Prehybridization:
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Place embryos in 1 - 2 mls of Prehybridization solution (PHS)
Let the embryos settle to the bottom
Replace with fresh PHS and place at 70°C in preheated water bath for 2-3 hrs
Embryos can be stored at this stage at -20°C
Prehybridization Solution (PHS)
Make up in a 50ml falcon
25mls Formamide
12.5mls 20x SSC
460ul 1M Citric Acid
500ul Tween-20
Bring up to 50mls with DEPC-HOH
(can be stored at -20°C)
Hybridization:
 Replace PHS with 70°C Hybridization solution (HS)
 Add 3ul of probe for every 1ml of HS
 Incubate o/n at 70°C in the water bath
Hybridization Solution (HS):
Make up in a 50ml falcon
25mls Formamide
12.5mls 20x SSC
460ul 1M Citric Acid
125ul tRNA
100ul Heparin (50ug/ml)
500ul Tween-20
Bring up to 50mls with DEPC-HOH
(can be stored at -20°C)
Day 2
Washes:
 1 x 10 mins in 75% PHS/ 25% 2x SSC @ 70°C
 1 x 10 mins in 50% PHS/ 50% 2x SSC @ 70°C
 1 x 10 mins in 25% PHS/ 75% 2x SSC @ 70°C
 1 x 10 mins in 2x SSC @ 70°C
 1 x 30 mins in .2x SSC @ 68°C
 2 x 10 mins in MAB @ rt
2
MAB (Maleic Acid Buffer)
2L Stock
100mM Maleic Acid 23.2g
150mM NaCl 17.5g
NaOH 15g
D-HOH
pH to 7.5 (important)
Autoclave then add .1% Tween-20
Pre-block Embryos:
 Move embryos to a plate of the appropriate size
 Pre-block in 1- 2mls of blocking solution at rt while oscillating for 3+hrs
Blocking Solution: 1 part 10% BMB; 1 part heat deactivated lamb serum; 3 parts MAB
10% BMB
2g Boehringer blocking reagent
20mL MAB
Heat to 45°C until dissolved
Divide into 1ml aliquots
Lamb Serum
Heat to 55°C for 30mins to deactivate
Divide into 1ml aliquots
Pre-block a-DIG
 Simultaneously pre-block a-DIG in BS at rt while oscillating for 3+hrs 
 1:2000 dilution in blocking solution

Block Embryos
 Remove pre-block and replace with pre-blocked a-DIG solution
 Incubate at 4°C o/n while oscillating 
Day 3
Washes
 1 x 5 mins in MAB
 2 x 10 mins in MAB
 1 x 30 mins in MAB
 1 x 1 hr in MAB
 3 x 5 mins in AP buffer
AP Buffer (15ml)
900ul Tris
900ul NaCl
450ul MgCl2
16.2ul Tween-20
13.86ml DEPC-HOH
3
Stain Embryos
 Pull off excess AP Buffer and add 200ul – 1ml staining solution
Stain: 2.5ml AP + 8.75ul BCIP + 11.25ul NBT
 Wrap in foil to keep dark and oscillate at rt
 Check after 15mins and then every 30mins until sufficiently stained
 Wash 2x 5mins in PBSt to stop the reaction
Dehydrate Embryos
 1 x 10mins in 25% Methanol/ 75% PBSt
 2 x 10mins in 50% Methanol/ 50% PBSt
 Place in 100% Methanol o/n at 4°C

Day 4
Rehydrate Embryos
 2 x 10mins 50% Methanol/ 50% PBSt
 1 x 10mins 25% Methanol/ 75% PBSt
 2 x 10mins PBSt
Glycerol Series
 4hrs 25% glycerol/ 75% PBSt
 6hrs 50% glycerol/50% PBSt
 Store in 75% glycerol/ 25% PBSt
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Note: Glycerol Series facilitates clearing. Although shorter time at each stage will not hurt, the
longer they remain at each stage the more clear they will become.
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