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Whole Mount in Situ

Procedure

  1. Dissect embryos in PBS, remove membranes and dissect as necessary.

    For older embryos use the forceps and make a hole in the roof of the hind brain and open the forebrain.

  2. Fix in 4% paraformaldehyde 4°C O/N, rocking in 5-10 ml volume.
  3. Wash in PBT 2X for 5 minutes at 4°C, gently rocking.
  4. Wash in sequence in 25%, 50%, 75% methanol:PBT for 10 minutes at RT, rocking.
  5. Wash 2X in 100% methanol for 10 min, rocking.
  6. Store in 100% methanol at -20°C.

DIG-Labeled Probe Synthesis

Linearizing Plasmid

Reaction Mix

  • 10X EZ Buffer™: 2.0 µl
  • Reaction EZ (20 U): 1.5 µl
  • Plasmid DNA 1 µg/µl: 5.0 µl
  • H2O: 12.5 µl
  1. Incubate 37°C for 1-3 hours. Run 1 µl on 1% TBE gel to check for complete digestion.
  2. Add 80 µl TE8 buffer, extract with 100 µl phenol:chloroform, spin 5 minutes at RT and transfer top aqueous layer to new tube.
  3. Repeat extraction. Then extract once with 100 µl chloroform and save aqueous layer.
  4. Add 1/10 volume 3M sodium acetate, 2 volumes ethanol, 1 µl glycogen or yeast tRNA. Incubate for 1 hour at -20°C.
  5. Spin 15 minutes at 4°C, maximum speed. Remove supernatant and wash pellet with 70% ethanol. Air dry.
  6. Resuspend pellet in 10 µl TE8 (0.5 µg/µl) buffer and store at 4°C.

Reaction

  • DEPC-H2O: 12.5 µl
  • 10X Transcription Buffer: 2.0 µl
  • 2.0 µl DIG Nucleotide Mix:

    10mM G, A, CTP
    6.5mM UTP
    3.5mM DIG-UTP:

  • Linearized Plasmid 0.5 µg/µl: 2.0 µl
  • Polymerase (T7, T3): 1.0 µl
  • RNase Inhibitor: 0.5 µl
  • Exception:
    When using SP6 polymerase, adjust reaction to:

    4 µl 5X Transcription Buffer
    2.0 µl 100mM DTT
    9.5 µl DEPC-H2O

Run-Off Transcription

  1. Incubate 37°C for 2 hours.
  2. Check transcription by running 1 µl on 1% TBE gel.

    A single predominant band should be seen at a greater intensity than the plasmid band. Additional fainter higher MW bands may be present.

  3. Add 1 µl DNase (RNase-free) and incubate at 37°C for 15 minutes.
  4. Add 100 µl TE8, 10 µl 4M LiCl, 300 µl ethanol and incubate at -20°C for 1 hour to O/N.
  5. Spin 10 minutes in microfuge at 4°C, maximum speed. Wash pellet with 70% ethanol and air dry pellet.
  6. Redissolve pellet in 50 µl TE8, and add 50 µl hybridization buffer. This can be stored at -20°C for several months.

Notes

  • CsCl DNA works best, or miniprep DNA that has not been RNased
  • Enzymes which leave 3' overhangs should not be used for run-off transcription

Day 1: Pretreament and Hybridization -- ~5 hours

  • All steps are performed on nutator
  • All washes are performed in glass scintillation vials in volumes of 5-10 mls, unless otherwise specified
  • Make all solutions fresh with DEPC-H2O and filter
  1. Transfer emrbyos to clean scintillation vials and rehydrate in 75%, 50%, 25% methanol:PBT-DEPC.
  2. Wash 2X in PBT-DEPC, 5 minutes each.
  3. Bleach embryos with 6% H2O2 in PBT for 1 hour at RT.
  4. Wash 3X PBT for 5 minutes at RT.
  5. Treat with proteinase K in PBT for 15 minutes at RT.

    For E8.0-9.0, use 0.333-0.25 µg/ml (1/30-1/40 dilution of 10 µg/ml)

    E9.5-11.5, use 0.5-2.0 µg/ml (1/20-1/5 dilution of 10 µg/ml)

  6. Wash with 2 mg/ml glycine in PBT for 10 minutes at RT.

    Make fresh and filter.

  7. Wash 2X PBT for 3 minutes at RT.
  8. Postfix with 4% paraformaldehyde and 0.2% glutaraldehyde for 20 minutes at RT.
  9. Wash 2X with PBT at RT.

    Transfer to 1.5-ml o-ring screw-top tubes. For larger embryos, leave in scintillation vials and adjust volumes as necessary, but be sure not to invert tubes, as embryos may stick to the cap after this point.

  10. Add 1.0 ml prehyb to tubes and mix gently by inversion. Remove and add another ml. Prehyb at 70°C for 1 hour.

    At this point, while still in prehyb, you can store the embryos at -20°C, either before or after heating.

  11. Make up hyb solution (1 ml prehyb + approx 1 µg/µl DIG-probe [10-25 µl usually]), and keep liquid at 37°C until ready to add to embryo.
  12. Remove prehyb and add 0.5 ml hyb solution. Invert gently several times, and then remove liquid. Add final 0.5 ml hyb solution and hybridize O/N at 70°C.

    Note
    :
    If you keep your formamide frozen, take it out of freezer to thaw O/N so can be used to make solutions for Day 2.

Day 2: Posthybridization and Antibody (Ab) Hybridization -- ~7 hours

Embryos are extremely sticky in Solutions I and III. Be very careful not to touch the embryos with pipette tip or plastic pipette once in these solutions!!

  1. Add 5 ml Solution I to clean glass scintillation vials. Transfer embryos from Eppendorf tubes into pre-warmed Solution I in scintillation vials.
  2. Wash 3X for 30 minutes in Solution I at 70°C.
  3. Switch to pre-warmed Solution III and wash 2X for 30 minutes at 65°C.
  4. Wash with fresh TBST (0.1% Tween 20 and 2mM levimasole) 3X for 5 minutes each at RT. Then transfer embryos to o-ring capped screw tubes during last wash.
  5. Preblock with 1 ml 10% sheep serum (heat-inactivated) in TBST for 2.5 hour at RT, rocking.
  6. While preblocking, adsorb secondary antibody*.
  7. Remove blocking serum and add 1 ml Ab mixture. Invert gently several times then remove. Add other ml of Ab mixture and rock gently O/N at 4°C.

*Adsorb Secondary Antibody

Per embryo (2 ml final volume per embryo final):

  • Put approx 3 mg (per embryo) embryo powder in a screw capped tube
    Scale up per embryo
  • Add 0.5 ml TBST (per 2 ml final) and rock at 70°C for 30 minutes
  • Vortex for 10 minutes
  • Cool on ice for 5 minutes, and add 5 µl sheep serum (heat-inactivated) and 1 µl mouse β-DIG (per 2 ml final)
  • Shake gently at 4°C for 1 hour.
  • Spin at 4000 rpm for 10 minutes at 4°C
  • Collect supernatant into a common 15-ml tube
  • Dilute with 1% sheep serum (heat-inactivated) in TBST to a final volume of 2 ml/embryo (~1.5 ml per embryo)
  • Spin mixture again at 4000 rpm for 10 minutes at 4°C

Day 3: Post-Ab Hybridization -- ~6 hours

  1. Transfer embryos from o-ring tubes into 5 ml fresh TBST with 2mM levimasole in glass scintillation vials. Wash 3X for 5 minutes each at RT.
  2. Wash 5X 1-1.5 hour each in TBST with 2mM levimasole at RT, rocking.
  3. Wash O/N in TBST with 2mM levimasole, rocking, at 4°C.

Day 4: Detecting -- ~1-2 hours

  1. Wash 3X in NTMT 1 minute at RT.
  2. Transfer embryos to o-ring tubes during last NTMT wash.
  3. Prepare Reaction Mix**.
  4. Remove NTMT.
  5. Add 1 ml Reaction Mix, and mix gently. Remove and add new ml of Reaction Mix.
  6. Cover tubes with foil and place on rocker for 20 minutes at RT. Check embryos to see if you are getting a signal.

    If not developed by 20 minutes, monitor ~once an hour, or as you feel needed. After each check, add fresh reaction mix.

  7. When the reaction is judged complete, transfer to scintillation vials and wash 2X with NTMT for 10 minutes each at RT, covered in foil.
  8. Wash 2X in PBT for 10 minutes at RT.

    If probe gives you high background, you can extend this step at 4°C for several days.


  9. Post fix with 4% paraformaldehyde/0.1% glutaraldehyde for 1 hour at RT. Wash 2X PBT. Store at 4°C in Eppendorf tubes for several months. Take pictures as soon as possible after development since stain will fade.
  10. To clear embryos to make visualization of light staining easier:

    Wash embryos with increasing concentrations of glycerol in PBT: 30%, 50%, 70%, 80% for 15 minutes to 1 hour, depending on embryo size. You can add 0.05% azide for long term storage at 4°C.

**Reaction Mix

  • 6.75 µl NBT
  • 5.25 µl BCIP
  • 2 ml NTMT

    Use fresh stocks of NBT [0.075 g/ml 70% dimethylformamide (DMF)] and BCIP-Na salt (0.05 g/ml H2O [or BCIP-toluidine salt (0.05 g/ml DMF)].

Solutions

BCIP

  • 0.05 g per 1 ml H2O for sodium salt
    or

  • 1 ml DMF for toluidine salt

DEPC-H2O

  • 0.1% DEPC in H2O
  • Let sit at 37°C O/N
  • Autoclave

Embryo Powder

  • Sacrifice 1-2 litters of E12.5-E14.5 mouse embryos

    One E13.5 litter should yield 200-400 mg embryo powder

  • Remove membranes, and homogenize in minimum volume ice-cold PBS. It is preferable to mush embryos up in PBS using mortar and pestle, then homogenizing by pulling back and forth through an 18g needle
  • Add 4 volumes ice-cold acetone
  • Vortex and put on ice for 30 minutes
  • Spin down in Oak Ridge tubes at 10,000 g for 10 minutes at 4°C
  • Discard floating debris and rinse pellet in ice-cold acetone
  • Discard floating debris and rinse pellet in ice-cold acetone
  • Allow pellet to completely dry on filter paper
  • Grind to powder with mortar and pestle
  • Store in Eppendorf tubes at -20°C

NBT

  • 0.075 g in 1 ml dimethylformamide (DMF)

NTMT

Make up fresh and filter before use.

Final Concentration

  • 100mM NaCl
  • 100mM Tris, pH 9.5
  • 50mM MgCl2
  • 0.1% Tween-20
  • 2mM Levamisole

For 100 ml

  • 2 ml 5M NaCl
  • 5 ml 2M Tris, pH 9.5
  • 5 ml 1M MgCl2
  • 0.1 g Tween-20
  • 0.048 g Levamisole
  • 88 ml H2O

4% Paraformaldehyde (PFA)

This is good for ~1 week, although fresh PFA is always best.

  • 4 g Paraformaldehyde
  • 90 ml H2O
  • 20 µl 10N NaOH
  • Mix and heat at 65°C for ~10 minutes. Swirl to ensure all of paraformaldehyde is in solution.
  • Add 10 ml 10X PBS for a final volume of 100 ml.
  • Filter and store at 4°C.

PBT

  • Make 10X PBS in DEPC-H2O
  • Autoclave.
  • Dilute to 1X with DEPC-H2O
  • Add 0.1% Tween-20
  • Filter before use

Prehybridization (Prehyb) and Hybridization (Hyb) Solutions

All components should be made with DEPC-H2O.

Heat to 65°C once made, then filter and store aliquoted at -20°C.

For hybridization solution, add approx 1 µg probe per ml prehyb solution.

Final Concentration

  • 50% Formamide
  • 5X SSC, pH 4.5
  • 50 µg/ml Yeast RNA
  • 1% SDS
  • 50 µg/ml Heparin

For 100 ml

  • 5 ml Formamide
  • 25 ml 20X SSC, pH 4.5
  • 0.5 ml 10 mg/ml Yeast RNA in DEPC-H2O
    (Extracted 2X with phenol:chloroform)

  • 10 ml 10% SDS
  • 0.1 ml 50 mg/ml in DEPC-H2O
  • 14.8 ml DEPC-H2O

Sheep Serum

  • Inactivate by heating to 55°C for 60 minutes
  • Can be stored in aliquots at -20°C

Solution I

Make up fresh. Preheat before filtering and before use.

Use approximately 25 ml/embryo.

Final Concentration

  • 50% Formamide
  • 5X SSC, pH 4.5
  • 1% SDS

For 100 ml

  • 50 ml Formamide
  • 25 ml 20X SSC, pH 4.5
  • 10 ml 10% SDS
  • 15 ml H2O

Solution III

Make up fresh. Preheat before filtering and before use.

Use approximately 25 ml/embryo.

Final Concentration

  • 50% Formamide
  • 2X SSC, pH 4.5

For 100 ml

  • 50 ml Formamide
  • 10 ml 20X SSC, pH 4.5
  • 40 ml H2O

20X SSC, pH 4.5

Final Concentration

  • 3M NaCl
  • 0.3M Na3-citrate
  • Adjust pH with 5M citric acid.

 

For 1 Liter

  • 175.3 g
  • 88.2 g
  • 800 ml H2O
  • Bring to 1 liter with H2O

TBST

Make from stock of 10X TBS, dilute to 1X and add Tween-20 and levimasole.

Does not require DEPC-H2O.

100 ml 10X TBS

  • 8 g NaCl
  • 0.2 g KCl
  • 25 ml 1M Tris, pH 7.5
  • Bring to 100 ml with H2O

100 ml TBST

  • 10 ml 10X TBS
  • 0.1% Tween-20
  • 2mM Levamisole
  • Bring to 100 ml with H2O

© 2014 The University of Texas MD Anderson Cancer Center