Whole Mount Fluorescence In Situ Hybridisation

Kindly provided by Dr Jonathan Rast, University of Toronto

Adapted from:

  • Ransick, A., Ernst, S., Britten, R.J. and Davidson, E.H. (1993) Whole Mount In Situ Hybridization Shows Endo 16 to be a Marker for the Vegetal Plate Territory in Sea Urchin Embryos. Mech. of Develop. 42: 117-124.
  • M. A. Harkey, H. R. Whiteley, A. H. Whiteley, Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis Mech. Dev., 37(3), 173-84 (1992)
  • Eric A. Gustafson and Gary M. WesselPolycomb group gene expression in the sea urchinDev Dyn. Jul 2008; 237(7): 1851–1861.

Solutions Needed:

  • DEPC dH2O
  • 1M MOPS pH 7.0 – Filter sterilize and store @ room temperature in the dark
  • Artificial sea water (ASW) – Recipe below
  • 16% paraformaldehyde (PFA; Electron Microscopy Sciences Cat. #15710 10x10ml)
  • 30%, 50% and 70% EtOH/DEPC H2O – Keep @ 4C
  • Deionized formamide1
  • 50% Polyethelene glycol (PEG – Sigma #P-5413)
  • 20mg/ml or 10mg/ml Yeast RNA2 – Keep @ -20C
  • 50X Denhardst solution – Keep @ -20C
  • 0.5M EDTA
  • 2M Tris pH 7.5
  • 1M Tris HCl pH 9.5
  • 1M MgCl2
  • 5M NaCl
  • 10% Tween 20
  • 20X, 1X and 0.1X SSC
  • Glycine Solution
  • 1% H2O2 – dilute from 30% stock (Sigma #216763) immediately before use
  • Heat inactivated sheep serum3
  • 100mM Levamisole – Keep @ -20C
  • Dimethylformamide (DMF)
  • 75mg/mL NBT in 70% DMF – Keep @ -20C in the dark
  • 50mg/mL INT in 100% DMF – Keep @ -20C in the dark
  • 50mg/mL BCIP in 100% DMF – Keep @ -20C in the dark
  • 15%, 30% and 50% glycerol

1 Add 5g of DOWEX ion-exchange resin (Sigma #428736-100G) to a new 500ml bottle of formamide (Bioshop #FOR001). Mix well and let the resin settle. Every once in a while, after using the formamide, mix the beads in and let settle again until next use.

2 Dissolve 20mg of yeast RNA (Sigma #R6625-25G) in 20ml dH2O, or as much as possible (there is some non-soluble material). Extract 2X with Phenol:chloroform :isoamyl alcohol. Extract 2X with chloroform. Precipitate by adding 0.1 volume of 3M NaAC pH 5.2 and 2 volumes of 100% EtOH, and store @ -20C O/N. Wash the pellet with 70% EtOH, dry and resuspend in 10ml nuclease-free water. Dilute 100X (1ml in 99ml dH2O) and measure concentration. The target concentration is 200ng/ml when diluted this way to give 20mg/ml stock. Adjust volume and measure concentration again as necessary. Aliquot in 500ml and 1ml volumes and store @ -20C.

3 Aliquot Sheep serum (Sigma #S3772-10ml) in 50ml to 750ml volumes. Incubate @ 56C (absolutely no higher than 60C!) for 30 minutes. Centrifuge @ max speed for 2 minutes and store upright @ -80C until frozen. Do not thaw aliquots more than twice.

Fixative 1 (for Larvae)

16% PFA 10 mL
1M MOPS 4 mL
5M Sodium chloride 4 mL
Water 22 mL
Total volume 40 mL

Fixative II (for embryos and early pluteus larvae)

16% PFA 10 mL
Artificial Sea Water (ASW) 13 mL
1M MOPS 1.3 mL
5M Sodium chloride 2.6 mL
Water 13.1 mL
Total volume 40 mL

MOPS Buffer

Tween 20 100 mL
1M MOPS 10 mL
5M Sodium chloride 10 mL
Water 80 mL
Total volume 100 mL

Artificial Sea Water

Sodium chloride/td> 28.32 g
Potassium chloride 0.77 g
Magnesium chloride hexahydrate 5.41 g
Magnesium sulphate septahydrate 7.13 g
Calcium chloride 1.18 g
Sodium hydrogen carbonate (bicarbonate) 0.2 g
Water To 1 L

TBS-TWEEN

2M Tris-HCL, pH7.5 15 mL
5M Sodium chloride 4.5 mL
10% Tween 20 1.5 mL
Water 129 mL
Total 150 mL

Hybridisation buffer

Deionised formamide 500 mL
50% Polyethylene glycol 200 mL
5M Sodium chloride 120 mL
20 mg/mL yeast tRNA 25 mL
50X Denhardt's reagent 20 mL
10% Tween 20 10 mL
0.5M EDTA 10 mL
Water 125 mL
2M Tris-HCl pH7.5 10 mL
Total 1 L

20X SSC

Sodium chloride 43.83 g
Sodium citrate 22.06 g
Water to 250 mL
  • pH to 7.5 with very dilute NaOH

Blocking buffer

TBS-Tween (TBST) 1.9 mL
Sheep serum 0.1 mL

Alkaline Phosphatase buffer

1M Tris-HCl pH9.5 0.5 mL
5M Sodium chloride 100 mL
1M Magnesium chloride 0.25 mL
10% Tween 20 50 mL
100 mM Levamisole 50 mL
Water 4.05 mL

TBSTE

TBS-Tween 15 mL
0.5M EDTA 30 mL

Staining Solution [purple]

AP buffer 900 mL
Dimethylformamide 100 mL
BCIP 3.5 mL
NBT 4.5 mL

Staining solution (yellow)

AP buffer 900 mL
Dimethylformamide 100 mL
BCIP 4 mL
INT 4 mL

Glycine Solution

  • 0.75g glycine in ~40ml dH2O
  • Adjust pH to 2.2 with HCl.
  • Add 50ml 10% Tween 20
  • Bring volume up to 100ml (check pH).
  • Filter and store @ room temperature.

Antibodies

  • AP conjugated anti-DIG – Roche # 11 093 274 910
  • AP conjugated anti-DNP – Mirus 6011 (purchase from Fisher Scientific)
  • POD-conjugated anti-DIG – Roche # 11 207 733 910
  • POD-conjugated anti-Flu – Roche # 11 426 346 910

Staining kit

  • TSA plus Cy3/Cy5 system – Perkin Elmer # NEL752001


Fixing

1. Fix ~150,000 embryos in ~4mL of fixing solution overnight at 4C.

Note:Fixing precious samples can be done in wells of a 96-well plate. Care should be taken to remove as much water as possible before adding fix. A “fix wash” or two can also be done, replacing with fresh fix soon after the first addition.

2. Wash 5X in cold MOPS buffer.

3. Dehydrate on ice with cold 30%, 50% and 70% EtOH/DEPC water. Simply let embryos settle or spin gently between washes.

4. Store at -20C in 70% EtOH/DEPC water.

Procedure – Day 1

Notes: As a rule of thumb, I use ~100-125ml of each solution, except for block and detection buffers, where I have uses less (as little as 50ml). Mix in each new solution carefully by swirling embryos.

1. Transfer up to ~ 200 embryos to a well of a 96-well plate.

2. Rehydrate embryos with 50% EtOH/DEPC H2O and then 30% EtOH/DEPC H2O. Alternatively, freshly fixed embryos can be used right away without dehydrating. Just wash 3X in MOPS buffer as above and proceed with following steps below.

3. Wash embryos 4X in TBST.

4. Transfer embryos to hybridization buffer in stages: 2:1, 1:2 and 0:3 ratios of TBST:hybridization buffer. These viscous solutions need extra swirling to mix them well.

5. Prehybridize for 1hr @ 65C.

6. Prepare hybridization buffer/probe mix (~30ng/mL) and heat @95C for 10 minutes. Cool on ice for 5-10min and warm @ 65C.

7. Remove as much of the pre-hyb buffer as possible and add the probe to each well. Mix well, cover with a piece of MicroSeal A film and hybridize O/N @ 65C.

Tip: Store TBST and 50:50 solution @ 65C O/N so they’ll be warm in the morning.


Procedure – Day 2

1. Wash embryos in 50:50 hybridization buffer:TBST 10 minutes @ 65C. Transfer the embryos to a new well; this helps reduce background.

2. Wash embryos 2X with TBST for 10 minutes each @ 65C.

3. Wash embryos 2X in 1X SSC and once in 0.1X SSC for 30 minutes each @ 65C.

4. Wash embryos 2X in TBST at room temperature.

5. Incubate embryos in blocking buffer for 30 minutes @ room temperature.

6. Incubate embryos in blocking buffer containing 1:2000 dilution of AP conjugated anti-DIG antibody for 1 hour @ room temperature.

7. Wash embryos 4X in TBST, for 7-10 minutes each.

8. Wash embryos 2X in AP buffer.

9. Stain embryos in fresh staining buffer [purple] for 20 minutes to overnight, in the dark, checking them regularly. Be careful when checking staining to avoid taking too long: this can cause background due to the light sensitivity of the staining solution.

10. Wash embryos 2X in TBSTE, 10 minutes each, to stop the staining reaction.

11. Transfer embryos to 15% then 30% and finally 50% glycerol. Glycerol further inhibits staining so embryos should be transferred to 30% glycerol on the same day. 50% glycerol transfer can be performed the following day if desired.

12. Cover with MicroSeal A film and store away from light.


General Considerations

  • I try to use the DIG probe for the “higher” expressing gene, and the DNP probe for the “lower” expressing gene whenever possible.
  • Staining with the more stable purple color first is preferred, followed by the yellow.

Procedure – Fixing and Day 1

Follow the procedure as for 1 color WMISH for fixing and day 1, except for hybridization (Steps 7&8). Add 30ng/ml of each of 2 probes, one labeled with DIG and one with DNP.

Procedure – Day 2

Follow Steps 1 through 9 as for 1 color WMISH (p. 4). It is recommended to use with the anti-DNP antibody first here in step 6 (1:2000 dilution in blocking buffer).

10. Wash embryos 2X in TBST

11. Wash embryos in Glycine solution for 10 min @ room temperature.

12. Wash embryos 2X in TBST

Procedure – Day 3

Note: If day 2 staining is fast for all samples, day 3 can be done the same day, but it is a very long day, so I usually stop before probing with the second antibody.

1. Incubate embryos in blocking buffer for 30 minutes @ room temperature.

2. Incubate embryos in blocking buffer containing 1:2000 dilution of AP conjugated anti-DIG antibody for 1 hour @ room temperature.

3. Wash embryos 4X in TBST, for 7-10 minutes each.

4. Wash embryos 2X in AP buffer.

5. Stain embryos in fresh staining buffer [yellow] for 20 minutes to overnight, in the dark, checking them regularly. Be careful when checking staining to avoid taking too long: this can cause background due to the light sensitivity of the staining solution.

6. Wash embryos 2X in TBSTE, 10 minutes each to stop the staining reaction.

7. Transfer embryos to 15% then 30% and finally 50% glycerol. Glycerol further inhibits staining so embryos should be transferred to 30% glycerol on the same day. 50% glycerol transfer can be performed the following day if desired.

8. Cover with MicroSeal A film and store away from light.


(Fluorescent variation)

General Considerations;

You need POD (HRP) conjugated antibodies to stain with TSA kit. Cy3 tends to have more background (probably due to the fixing of the embryos with PFA), so it’s best to stain the “higher” expressing gene with Cy3 and the “lower” expressing gene with Cy5. Alternatively, a Fluoroscein TSA kit is also available from Perkin Elmer.

Procedure – Fixing and Day 1

Follow the procedure as for 1 color WMISH for fixing and day 1, except for hybridization (Steps 7 & 8). Add 30ng/ml of each of 2 probes, one labeled with DIG and one with Flu.

Procedure – Day 2

Follow Steps 1 through 7 as for 1 color WMISH (p. 4). Use the POD conjugated anti-DIG antibody here in step 6 (1:2000 dilution in blocking buffer).

8. Stain 5-15 minutes with TSA kit Cy3 (or Cy5) diluted at 1:400 using diluent provided in the kit.

9. Wash embryos 4X in TBST. You can check staining under the microscope at this point and wash some more if needed.

10. Wash embryos in 1% H2O2 solution.

11. Wash embryos 1X in TBST.

12. Wash embryos in Glycine solution for 5 min @ room temperature.

13. Wash embryos 3X in TBST.

14. Incubate embryos in blocking buffer containing 1:2000 dilution of POD conjugated anti-Flu antibody for 1 hour @ room temperature.

15. Wash embryos 4X in TBST, for 7-10 minutes each.

16. Stain 5-15 minutes with TSA kit Cy5 (or Cy3) diluted at 1:400 using diluent provided in the kit.

17. Wash embryos 4X in TBST to stop the staining reaction. You can check staining under the microscope at this point and wash some more if needed.

18. Transfer embryos to 15% then 30% and finally 50% glycerol. Glycerol further inhibits staining so embryos should be transferred to 30% glycerol on the same day. 50% glycerol transfer can be performed the following day if desired.

19. Cover with MicroSeal A film and store away from light.

Sham Nair 2014