Whole-mount in situ hybridization (Conlon lab)

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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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The End