DIG In Situ Hybridization

 dsDNA PCR probe

Probe labeling

Hybridization buffer

Materials

Amount/100 ml

Final Conc.

dextran sulfate

10 g

10%

deionized formamide

50 ml

50%

20 ~SSC

10 ml

2 ~

0.1 M NaPB (pH7)

16 ml

16 mM

250 mM EDTA

400 ml

1 mM

50 ~ Denhardt's solution

2 ml

5 ~

10 mg/ml salmon sperm DNA

4 ml

400 mg/ml

10 mg/ml yeast tRNA

4 ml

400 mg/ml

10% N-lauroylsarcosine

10 ml

1%

Pretreatment

  1. 4% parafomaldehyde in 0.1 M PBS, 20 min
  2. PBS, 10 min
  3. PBS, 10 min
  4. 0.2 N HCl, 10 min
  5. 10 mg/ml Proteinase K in 1 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 5-30 min
  6. PBS, 10 min
  7. PBS, 10 min
  8. (Acetylate)
  9. DW
  10. Dehydrate (70-100% Ethanol)
  11. Chroloform, 10 min
  12. 100% Ethanol
  13. Air-dry

Hybridization

  1. Add the PCR-labeled DNA probe (2 ml in 1ml buffer) to the hybridization solution and mix thoroughly.
  2. Heat-denature the probe in hybridization solution by heating to >95ίC for 10 min and cool in an ice bath for 5 min.
  3. Pipette 100 ml of the probe solution onto clean coverslips taking care to avoid air bubbles.
  4. Cover with pre-treated sections on glass slide.
  5. Place slides in a humid chamber containing 2 ~ SSC, 50% formamide.
  6. Incubate overnight at 42ίC.

Washing

  1. 2 ~ SSC at room temperature until coverslips become detached.
  2. 2 ~ SSC 4 times for 15 min each at 42ίC.
  3. 0.2 ~ SSC for 15 min at 42ίC.

Visualization

  1. DIG buffer 1, 5 min
  2. DIG buffer 2 (1% blocking reagent in DIG buffer 1), 60 min
  3. 5,000 ~ diluted anti-DIG antibody in DIG buffer 2, 30 min, or 4ίC, O/N
  4. DIG buffer 1, 15 min, twice
  5. DIG buffer 3
  6. 4.5 ml/ml NBT, 3.5 ml/ml BCIP in DIG buffer 3, 15 min`3 days in dark
  7. 10 mM Tris-HCl, 1 mM EDTA, 5 min
  8. Cover with coverslips for observation

ssRNA probe

Probe labeling

Hybridization buffer

Pretreatment

Hybridization

Washing

Visualization

Northern Hybridization

dsDNA PCR probe

Probe labeling

Hybridization buffer

Materials

Amount for 1 ml

Final Conc.

1M church NaPB (pH 7.2)

50 ml

50 mM

deionized formamide

500 ml

50%

20 ~ SSPE

250 ml

5 ~

50 ~ Denhardtfs solution

100 ml

5 ~

10 mg/ml salmon sperm DNA

40 ml

400 mg/ml

25 mg/ml yeast tRNA

2 ml

50 mg/ml

10% N-lauroylsarcosine

10 ml

0.1%

SDS

70 mg

7%

DEPC-DW

4? ml

up to 1 ml

Prehybridization

  1. Incubate membrane with hybridization buffer without probe in sealed plastic bag at 50ίC over 1 hour.

Hybridization

  1. Heat-denature the labeled DNA by heating to 95ίC (or by boil) for 10 min and cool in an ice bath for 5 min.
  2. Add the labeled, denatured DNA probe (in 5 ml) to the hybridization solution to a concentration of 5`25 ng/ml and mix thoroughly.
  3. Pipette 100 ml of the probe solution into clean hybridization bag taking care to avoid air bubbles.
  4. Sheal the opened edge of the bag with heating sealer.
  5. Place the bag in a pre-heated water bath at 50ίC.
  6. Incubate overnight at 50ίC.

Washing

  1. Wash membrane with 2 ~ SSC, 0.1% SDS twice for 5 min each at room temperature.
  2. Wash membrane with 0.1 ~ SSC, 0.1% SDS twice for 15 min each at 68ίC.

Visualization

  1. DIG buffer 1, 5 min
  2. DIG buffer 2 (1% blocking reagent in DIG buffer 1), 60 min
  3. 5,000`10,000 ~ diluted anti-DIG antibody in DIG buffer 2, 30 min, or 4ίC, O/N
  4. DIG buffer 1, twice, 15 min each
  5. DIG buffer 3
  6. 4.5 ml/ml NBT, 3.5 ml/ml BCIP in DIG buffer 3, 15 min`3 days in dark
  7. 10 mM Tris-HCl, 1 mM EDTA, 5 min
  8. Cover with coverslips for observation
See Roche Diagnostics Molecular Biochemicals, Guide Books for DIG-System