Molecular Techniques and Methods

In situ Hybridization to Metaphase Chromosomes

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

RNase A Solution (1 ml)
RNase A -------------------------------------------- 100 ug
2 x SSC --------------------------------------------- 0.1 ml of 20 x SSC
Distilled H2O ---------------------------------------- 0.9 ml


Pepsin Solution (1 ml)
0.005-0.02% Pepsin -------------------------------- 5-20 ul of 1% Pepsin
10 mM HCl ----------------------------------------- 1 ul of 11.6 M HCl
Distilled H2O ---------------------------------------- 1 ml


Post-Fix Solution (100 ml)
PBS ------------------------------------------------ 92.3 ml
50 mM MgCl2 -------------------------------------- 5 ml of 1 M MgCl2
1% Formaldehyde ---------------------------------- 2.7 ml of 37% Formaldehyde


Hybridization Solution-1 (10 ml)
60% Deionized Formamide -------------------------- 6 ml of 100% Deionized Formamide
2 x SSC --------------------------------------------- 1 ml of 20 x SSC
50 mM Sodium Phosphate Buffer (pH 7.0) ---------- 0.5 ml of 1 M Na-Phosphate Buffer
Distilled H2O ---------------------------------------- 2.5 ml


Washing Solution-1 (10 ml)
2 x SSC -------------------------------------------- 1 ml of 20 x SSC
60% Deionized Formamide ------------------------- 6 ml of 100% Deionized Formamide
Distilled H2O ---------------------------------------- 3 ml


Hybridization Solution-2 (10 ml)
50% Deionized Formamide ------------------------- 5 ml of 100% Deionized Formamide
2 x SSC -------------------------------------------- 1 ml of 20 x SSC
10% Dextran Sulfate -------------------------------- 1 g
50 mM Sodium Phosphate Buffer (pH 7.0) ---------- 0.5 ml of 1 M Na-Phosphate Buffer
Add distilled H2O to make a final volume of ------- 10 ml


Washing Solution-2 (10 ml)
2 x SSC --------------------------------------------- 1 ml of 20 x SSC
50% Deionized Formamide -------------------------- 5 ml of 100% Deionized Formamide
Distilled H2O ---------------------------------------- 4 ml


Hybridization Solution-3 (10 ml)
70% Deionized Formamide -------------------------- 7 ml of 100% Deionized Formamide
2 x SSC --------------------------------------------- 1 ml of 20 x SSC
50 mM Sodium Phosphate Buffer (pH 7.0) ---------- 0.5 ml of 1 M Na-Phosphate Buffer
Distilled H2O ---------------------------------------- 1.5 ml


TNT Buffer (10 ml)
10 mM Tris-HCl (pH 7.5) -------------------------- 0.1 ml of 1 M Tris-HCl
150 mM NaCl --------------------------------------- 0.3 ml of 5 M NaCl
0.05% Tween 20 ------------------------------------ 50 ul of 10% Tween 20
Distilled H2O ---------------------------------------- 9.55 ml


TNB Buffer (10 ml)
100 mM Tris-HCl (pH 7.5) -------------------------- 1 ml of 1 M Tris-HCl
150 mM NaCl --------------------------------------- 0.3 ml of 5 M NaCl
0.5% Blocking Reagent ------------------------------ 0.5 ml of 10% Blocking Reagent
Distilled H2O ---------------------------------------- 8.65 ml


Antibody 1 Solution (1ml)
Mouse monoclonal anti-DIG ----------------------------- 0.5 ug
TNB Buffer ---------------------------------------------- 1 ml


Antibody 2 Solution (1 ml)
DIG-conjugated sheep anti-mouse Ig --------------------- 2 ug
TNB Buffer ---------------------------------------------- 1 ml


Antibody 3 Solution (1 ml)
Fluorescein- or rhodamine-conjugated sheep anti-DIG -----2 ug
TNB Buffer ----------------------------------------------- 1 ml




PROCEDURES

Pretreatment of Metaphase Spreads of Chromosomal DNA on Slides

1. Incubate metaphase chromosome spreads with 100 ul of RNase A Solution under a coverslip for 1 hour at 37oC.

2. Wash slides for 5 min with 2 x SSC. Repeat 3 times.

3. Dehydrate slides in an ethanol series.

Ethanol
Time
25%
15 min
50%
15 min
75%
15 min
100%
15 min


4. Incubate slides with Pepsin Solution for 10 min at 37oC.

5. Wash slides with PBS for 5 min. Repeat

6. Wash slides with PBS containing 50 mM MgCl2.

7. Post-fix slides for 10 min at room temperature with a Post-Fix Solution.

8. Wash slides with PBS and dehydrate in an ethanol series.

Ethanol
Time
25%
15 min
50%
15 min
75%
15 min
100%
15 min



Denaturation and Hybridization on Slides

  • For denaturation and hybridization three alternative protocols are given.

    (a) For Probes Recognizing Repetitive Sequences in the Chromosomes

    9. Label 1 ug of DNA probe using digoxigenin (DIG)- or biotin-labeled nucleotide.

    10. Precipitate the labeled probe with 2.5 vol. of ethanol and 0.1 vol. of 3 M Sodium acetate.

    11. Resuspend the precipitated probe at a concentration of 10 ng/ul in Hybridization Solution-1.

    12. Incubate the tube for 15 min at 37oC with occasional vortexing until the precipitated probe DNA dissolves.

    13. Dilute the probe from the stock to the desired concentration (typically 2 ng/ul) in Hybridization Solution-1.

    14. Apply 5 ul diluted probe under a coverslip on the object slide and place the slide in an oven at 80oC for 2-4 min to denature the probe and target.
  • Sealing coverslip with rubber cement is not necessary.

    15. To hybridize, place the slides in a moist chamber at 37oC overnight.

    16. Wash slides for 5 min at 37oC with Washing Solution-1. Repeat 3 times.

    17. Wash slides for 5 min with the buffer to be used for immunological detection.


    (b) For Probes Recognizing Unique Sequences in the Chromosomes

    9. Label 1 ug of DNA probe using digoxigenin (DIG)- or biotin-labeled nucleotide.

    10. Precipitate the labeled probe with 2.5 vol. of ethanol and 0.1 vol. of 3 M Sodium acetate.

    11. Resuspend the precipitated probe at a concentration of 10 ng/ul in Hybridization Solution-2.

    12. Incubate the tube for 15 min at 37oC with occasional vortexing until the precipitated DNA dissolves.

    13. Dilute the probe from the stock to the desired concentration (typically 2 ng/ul) in Hybridization Solution-2.

    14. Apply 10 ul of the diluted probe solution under a coverslip on the object slide.

    15. Seal coversilp to slide with rubber cement.

    16. Place the slide in an oven at 80oC for 2-4 min.

    17. To hybridize, place the slides in a moist chamber at 37oC overnight.

    18. Wash slides for 5 min at 45oC with Washing Solution-2. Repeat 3 times.

    19. Wash slides for 2 min with 2 x SSC. Repeat 5 times.

    20. Wash slides for 5 min with the buffer used in detection.


    (c) Competition Hybridization: For Probes or Probe Cocktails Which Contain Repetitive Sequences Occurring throughout the Genome (e.g., Alu sequences)

    9. Label 1 ug of DNA probe using digoxigenin (DIG)- or biotin-labeled nucleotide.

    10. Precipitate the labeled probe with a 50-fold excess of yeast tRNA, 1/10th volume of 3 M Sodium acetate (pH 5.6), and 2.5 volumes of cold (-20oC) ethanol.

    11. Resuspend the precipitated probe at a concentration of 10-40 ng/ul in Hybridization Solution-2.

    12. Incubate the tube for 15 min at 37oC with occasional vortexing until the precipitated DNA dissolves.

    13. For hybridization, dilute the probe from the stock to the desired concentration in Hybridization Solution-2
  • For hybridization, use 2-5 ng/ul of cosmid probes, 10-20 ng/ul of chromosome specific libraries, or 20-40 ng/ul of YAC probes.

    14. Denature the probe at 75oC for 5 min, chill on ice, and allow annealing of the repetitive elements at 37oC for 1-2 hours.

    15. Prepare the chromosomes on the slide by adding Hybridization Solution-3.
  • Cover with a coverslip.

    16. Incubate in an oven at 80oC for 2-4 min to denature the chromosomes.
  • Remove the coverslip and quench the chromosomes in chilled 70% ethanol.
  • Dehydrate the slide by passing it through 90% ethanol, then 100% ethanol.

    17. Air dry the slide, preferably on a metal plate at 37oC.

    18. Hybridize the chromosomes overnight with 10 ul of the pre-annealed probe under a sealed coverslip.

    19. Wash slides for 5 min at 45oC with Washing Solution-2. Repeat 3 times.

    20. Wash slides for 5 min at 60oC with 0.1 x SSC. Repeat 3 times.

    21. Wash slides for 5 min with the buffer to be used in immunological detection.



    Single Color Fluorescent Detection with Immunological Amplification

    (a) For Biotin-Labeled Probe (Low Sensitivity)

    22. Wash slides briefly with TNT Buffer.

    21. Incubate slides for 30 min at 37oC with TNB buffer.

    22. Incubate slides for 30 min at 37oC with the proper dilution (typically 10 ug/ml) of a fluorescently labeled streptavidin-conjugate in TNB Buffer.

    23. Wash slides (3 x 5 min) with TNT Buffer.

    24. Dehydrate slides through an ethanol series.

    Ethanol
    Time
    70%
    5 min
    90%
    5 min
    100%
    5 min

  • Air dry slides.

    25. Stain with the appropriate DNA counterstain.

    26. Embed in Vectashield.

    27. View the slides by fluorescence microscopy, using appropriate filters.


    (b) For Biotin-Labeled Probe (High Sensitivity)

    22. Wash slides briefly with TNT Buffer.

    23. Incubate slides for 30 min at 37oC with TNB Buffer.

    24. Incubate slides for 30 min at 37oC with the proper dilution (typically 10 ug/ml) of a fluorescently labeled streptavidin-conjugate in TNB Buffer.

    25. Wash slides (3 x 5 min) with TNT Buffer.

    26. Incubate slides for 30 min at 37oC with the proper dilution of biotinylated goat anti-streptavidin (5 ug/ml) in TNB Buffer.

    27. Wash slides (3 x 5 min) with TNT Buffer.

    28. Incubate slides for 30 min at 37oC with the proper dilution (typically 10 ug/ml) of a fluorescently labeled streptavidin-conjugate in TNB Buffer.

    29. Wash slides (3 x 5 min) with TNT Buffer.

    30. Dehydrate slides through an ethanol series.

    Ethanol
    Time
    70%
    5 min
    90%
    5 min
    100%
    5 min

  • Air dry slides.

    31. Stain with the appropriate DNA counterstain.

    32. Embed in Vectashield.

    33. View the slides by fluorescence microscopy, using appropriate filters.


    (c) Digoxigenin-Labeled Probe (Low Sensitivity)

    22. Wash slides briefly with TNT Buffer.

    23. Incubate slides for 30 min at 37oC with TNB Buffer.

    24. Incubate slies for 30 min at 37oC with the proper dilution (typically 2 ug/ml in TNB Buffer) of a fluorescently labeled sheep anti-DIG antibody.

    25. Wash slides for 5 min with TNT Buffer. Repeat 3 times.

    26. Dehydrate slides through an ethanol series.

    Ethanol
    Time
    70%
    5 min
    90%
    5 min
    100%
    5 min

  • Air dry slides.

    27. Stain with the appropriate DNA counterstain.

    28. Embed in Vectashield.

    29. View the slides by fluorescence microscopy, using appropriate filters.


    (d) Digoxigenin-Labeled Probe (High Sensitivity)

    22. Wash slides briefly with TNT Buffer.

    23. Pipette 100 ul of TNB Buffer onto each slide and cover with a 24 x 50 mm coverslip.

    24. Incubate slides for 30 min at 37oC in a moist chamber (1 L beaker containing moistened tissues, covered with aluminum foil).

    25. Immerse the slides for 5 min in TNT Buffer to loosen the coverslips.

    26. Prepare fresh working solutions of three antibodies: Antibody 1, Antibody 2, and Antibody 3 Solutions.

    27. Pipette 100 ul of Antibody 1 Solution onto each slide and cover with a 24 x 50 mm coverslip.

    28. Incubate slids for 30 min at 37oC in a moist chamber.

    29. Wash slides (3 x 5 min) at room temperature with TNT Buffer.

    30. Pipette 100 ul of Antibody 2 Solution onto each slide and add a 24 x 50 mm coverslip.

    31. Incubate for 30 min at 37oC in a moist chamber.

    32. Repeat the washes as in Step 6.

    33. Pipette 100 ul of Antibody 3 Solution onto each slide and add a 24 x 50 mm coverslip.

    34. Incubate for 30 min at 37oC in a moist chamber.

    35. Dehydrate slides through an ethanol series.

    Ethanol
    Time
    70%
    5 min
    90%
    5 min
    100%
    5 min

  • Air dry slides.

    36. Stain with the appropriate DNA counterstain.

    37. Embed in Vectashield.

    38. View the slides by fluorescence microscopy, using appropriate filters.


    NOTES



    KIT INFORMATION


    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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