Whole-mount in situ hybridization (Conlon lab)
Protocol submitted by VGP from Conlon lab protocols [1]
link to protocol document [2]
Whole-Mount in situ Hybridization Protocol
Updated 10/2/2010 PT
Day 0 (Preparation of Digoxigenin-labeled probes)
Linearize DNA with enzyme (5ug max, approx 3-4ul of ‘dirty’ miniprep). Check on gel. Clean using ph/chl extraction or using Probequant G50 microcolumns (GE Healthcare): place column in tube, break bottom seal and loosen lid slightly, spin 1 min 750g, discard tube. Place column in fresh epp, pipette 50ul (min vol!) rxn onto centre of compact bead column, spin 2mins 750g = clean linear DNA.
Reaction:
Linear DNA ~250-500 ng (5ul linear DNA from Probequant column as above)
5X Promega Transcription buffer (10x if using NEB) 4 μl
DIG RNA Labeling Mix (UTP), Roche 1277073 2 μl
DTT (omit if using NEB buffer) 2 μl
RNase Inhibitor (RNasIn) 1 μl
RNA Polymerase (T7, T3, or SP6) 1 μl
H2O to 20 μl total
- 3-4 hr @ 37°C.
DNase Digestion (optional)
reaction 20 μl
10X RQ1 DNase Buffer 3 μl
RQ1 RNase-free DNase 7 μl
Total 30 μl
- 1 hr @ 37°C
Probe Purification
- Either repeat Probequant spin (make up rxn to 50ul before) or …
- Use QIAgen RNeasy Kit:
- Make up rxn to 100ul with RNAse free water
- Add 350 μl Buffer RLT
- Add 250 μl 100% EtOH
- Apply to column, spin 10K 15 sec.
- Transfer column to new tube
- Add 500 μl Buffer RPE
- Spin 15 sec, discard flow-through
- Add 500 μl Buffer RPE
- Spin 15 sec, discard flow-through
- Transfer column to new tube
- Spin 1 min.
- Add 45 μl DEPC-H2O to column
- Spin 1 min.
- Add flow-through BACK TO COLUMN
- Spin 1 minute
- Check 2 μl on agarose gel
Day 1 (Prehybridization)
Rehydration of embryos:
- Embryos should have previously been fixed in MEMFA for 2 hr @ RT or ON 4oC rocking, and stored @ -20oC MeOH (preferably ON before proceeding to enhance permeabilization)
MEMFA
MEM salts (10X) 1 ml
Formaldehyde (37%) 1 ml
DEPC-H2O to 10 ml
10X MEM Salts
MOPS 1 M 104.5 g
EGTA 20 mM 3.8 g
MgSO4 10 mM 1.24 g
DEPC-H2O to 500 ml
- Replace MeOH
- 5 min @ RT, replace 0.5 volume with DEPC-H2O. Allow embryos to settle before beginning timer.
- 5 min @ RT, replace 0.5 volume with DEPC-H2O
- 5 min @ RT, replace 0.5 volume with DEPC-H2O
- Replace all liquid w/ PBST
DEPC-H2O
DEPC 1 ml
H2O 1 L
(stir ON @ RT, then autoclave)
PBST
1X PBS (made w/ DEPC-H2O)
0.1% Tween 20
20X PBS
NaCl 2.74 M 160 g
KCl 54 mM 4.02 g
KH2PO4 30 mM 4 g
Na2HPO4 130 mM 18.46 g
DEPC-H2O to 1 L
(pH to 7.5 with HCL - autoclave)
Transfer to PBST: (wash preferably in >3ml rocking on side)
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
Permeabilization of Embryos/Removal of Vitelline Membrane:
For embryos hatched or with vitelline membrane removed
- 15 min @ RT in Proteinase K (extend to 20-25mins for older >st40)
Proteinase K (Sigma P-2308) – resuspend stock (18ug/ul) to 10 μg/μl. Add to embryos @ final concentration = 5 μg/ml (0.5 ul per ml PBST)
For early embryos still in vitelline membrane (digests membrane)
- 10 min @ RT in Proteinase K, Collagenase A, an Hyaluronidase. The membranes will come off during digestion or in the subsequent washes.
Proteinase K (Sigma P-2308) – resuspend stock to 10 μg/μl
Add to embryos @ final concentration = 10 μg/ml
Collagenase A (Roche 103 578) – resuspend stock to 1 mg/10 μl
Add to embryos @ final concentration = 2 mg/ml
Hyaluronidase (Sigma H-4272) – resuspend stock to 20 U/μl
Add to embryos @ final concentration = 20 U/ml
Washes to Remove Proteinase K:
- 5 min @ RT in PBST gently
- 5 min @ RT in PBST
- 5 min @ RT in PBST
Washes:
- 5 min @ RT in 1ml 0.1M Triethanolamine
0.1 M Triethanolamine
0.93 g Triethanolamine
30 μl 10 N NaOH (same as 10 M NaOH)
50 mL DEPC-H2O
- 5 min @ RT in 1ml 0.1 M Triethanolamine (2 mls per vial - DON’T REMOVE THIS SOLUTION)
Neutralization of free amines: (prevents electrostatic interaction between probe and basic proteins)
- 5 min @ RT, add 5 μl Acetic Acid Anhydride
- 5 min @ RT, add 5 μl Acetic Acid Anhydride
Washes:
- 5 min @ RT in PBST
- 5 min @ RT in PBST
Refixation:
- 20-30 min @ RT in 4% Paraformaldehyde/PBST
4% Paraformaldehyde
paraformaldehyde 2 g
10 N NaOH 1-2 drops
microwave a few seconds to dissolve
20X PBS (made w/ DEPC-H2O) 2.5 ml
Tween 20 50 μl
DEPC-H2O to 50 ml
Washes:
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5 min @ RT in PBST
- 5mins @ RT in 1:1 hyb:PBST
Transfer to Hybridization Buffer
- Add 1ml Hyb buffer.
Hybridization Buffer (store -20oC)
Formamide 25 ml
20X SSC (made w/ DEPC-H2O) 12.5 ml
Yeast Torula RNA 50 mg
Heparin 5 mg
50X Denhardt’s 1 ml
Tween 20 50 μl
CHAPS 50 mg
0.5 M EDTA 0.5 ml
DEPC-H2O to 50 ml
(store @ 4°C for up to a few days)
50X Denhardt’s
Ficoll Type 400 1 g
Polyvinylpyrrolidone 1 g
BSA Fraction V 1 g
DEPC-H2O to 1 L
(store @ -20°C)
20X SSC
NaCl 3 M 175.3 g
Na Citrate 0.3 M 88.2 g
DEPC-H2O to 1 L
(pH to 7.6 with HCl - autoclave)
- ON @ 60°C (or appropriate temp) in Hybridization Buffer
Day 2 (Hybridization)
Hybridization:
- Add 1-4 μg DIG-labeled probe to 1 ml Hybridization Buffer (~10 ul per ml)
- denature probe/hyb 5 min @ 75°C
- aspirate pre-hyb buffer from embryos and add the 1 ml probe/hyb
- ON @ 60-63°C
Day 3 (Antibody Staining) –
- Pre-warm SSC and MAB buffers to 60oC
Removal of Probe:
- remove old probe – store @ -20/-80°C for re-use
- rinse w/ Hybridization Buffer
- 10 min @ 60°C in 1 ml Hybridization Buffer
SSC Washes:
- 20 min @ 60°C in 2X SSC
- 20 min @ 60°C in 2X SSC
- 20 min @ 60°C in 2X SSC
- 20 min @ 60°C in 2X SSC
RNase Treatment: (optional – don’t do for low-expression genes)
- 30 min @ 37°C RNase
RNase
RNase A (4 mg/ml) 5 μl/ml (20 μg/ml total)
RNase T1 (20 U/μl) 0.5 μl (10 μ/ml total)
Washes: (unnecessary if RNase treatment was skipped)
- 10 min @ RT in 2X SSC
- 10 min @ RT in 2X SSC
High-Stringency Washes:
- 1 hr @ 60°C in 0.2X SSC
- repeat again for highly expressed RNAs or to decrease background staining (don’t perform for low expression RNAs)
Transfer to Maleic Acid Buffer:
- 15 min @ 60°C in 1X MAB
5X MAB
Maleinic Acid 0.5 M 58.04 g
NaCl 0.75 M 43.83 g
DEPC-H2O to 1 L
(pH to 7.5 w approx 9g NaOH pellets + additional NaOH solution)
- 15 min @ RT in 1X MAB
Blocking:
- 60 min @ RT in 1 ml Blocking Buffer
Blocking Buffer
1X MAB 80% 8 ml
Heat-inactivated lamb serum 20% 2 ml
BMBR 2% 0.2 g
Total 10 ml
To heat-inactivate lamb serum: 1 hr @ 60°C, spin 15 min @ 14,000 RPM, store @ -20°C (SPIN BEFORE USE ABOVE)
Antibody Incubation: (Fab Fragments, 1/2000 dil. Anti DIG AP Fab Fragments Roche 1093274)
- Add 0.5ul/ml antibody to fresh blocking buffer.
- Remove old blocking buffer, add antibody+blocking buffer onto embryos.
- ON @ 4° C (rocking + standing).
Day 4 (Removal of Antibody)
Washes:
- rinse in 1X MAB
- 1 hr @ RT in 1X MAB
- 1 hr @ RT in 1X MAB
- 1 hr @ RT in 1X MAB
- 1 hr @ RT in 1X MAB
- ON @ 4° in 1X MAB (repeat washes if background high)
Day 5 (Alkaline Phosphatase Staining)
Transfer to Alkaline Phosphatase Buffer:
- 5 min @ RT in AP Buffer
AP Buffer
1 M Tris pH 9.5 100 mM 5 ml
1 M MgCl2 50 mM 2.5 ml
0.5 M NaCl 100 mM 10 ml
Tween 20 0.1% 50 μl
Levamisol 5 mM 60 mg
ddH2O to 50 ml
- 5 min @ RT in AP Buffer
Staining:
- Add 1-2 ml BM Purple AP Substrate (Roche 1442074)
- KEEP IN THE DARK UNTIL BLEACHING STEP BELOW TO AVOID BACKGROUND STAINING!!
- Let stain anywhere from 1 to 8 hours until stain is at desired level
- Rinse with PBS
- 5 min @ RT in PBS
- 5 min @ RT in PBS
- 5 min @ RT in PBS
- 1hr @ RT in MEMFA
- 1 hr in MeOH (can be stored @ 4°C here)
Bleaching:
(can be done prior to Day 1 although requires re-fixing in MEMFA and starting from scratch. Also PFA fixation after Prot K treatment may need to be extended to 40mins/1hr as embryos are much more delicate.)
- ON @ 4°C in 5:1 MeOH/H2O2 (30% solution) under high light (with rocking and on a reflective background such as aluminum foil)
- Transfer and store in MeOH
Clearing:
- If you wish to clear embryos for image-taking, clear in 1:2 Benzyl Alcohol/Benzyl Benzoate (which can be REUSED!), but embryos MUST be fully dehydrated in MeOH before placement in BA/BB
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