Molecular Techniques and Methods

Detection of DNA Sequences in
Paraffin-Embedded Tissue Sections

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Proteinase K Solution (10 ug/ml)
Stock Proteinase K (20 mg/ml) ----------------- 5 ul
TES -------------------------------------------- 1 ml


TES (10 ml)
50 mM Tris-HCl (pH 7.4) ---------------------- 0.5 ml of 1 M Tris-HCl
10 mM EDTA ---------------------------------- 0.2 ml of 0.5 M EDTA
10 mM NaCl ----------------------------------- 0.02 ml of 5 M NaCl
Distilled H2O ----------------------------------- 9.28 ml


Silicone Solution (10 ml)
2% 3-Aminopropyltrithoxysilane ---------------- 0.2 ml
Acetone ---------------------------------------- 9.8 ml


Formaldehyde Fixatives (10 ml)
4% Formaldehyde ------------------------------ 0.11 ml of 37% Formaldehyde
0.1 M Sodium Phosphate Buffer ---------------- 1 ml of 1 M Sodium Phosphate Buffer
Distilled H2O ----------------------------------- 8.89 ml


Pretreatment Buffer (10 ml)
100 mM Tris-HCl ------------------------------ 1 ml of 1 M Tris-HCl
150 mM NaCl (pH 7.5) ------------------------ 0.3 ml of 5 M NaCl
Distilled H2O ----------------------------------- 8.7 ml


Blocking Buffer (10 ml)
0.5% Blocking Reagent ------------------------- 0.5 ml 10% Blocking Reagent
Pretreatment Buffer ----------------------------- 9.5 ml


Color Development Buffer (10 ml)
100 mM Tris-HCl ------------------------------ 1 ml of 1 M Tris-HCl
100 mM NaCl (pH 7.5) ------------------------ 0.2 ml of 5 M NaCl
50 mM MgCl2 --------------------------------- 0.5 ml of 1 M MgCl2
Distilled H2O ----------------------------------- 8.3 ml


Color Substrate Solution (5 ml)
NBT Solution ----------------------------------- 22.5 ul
BCIP (X-phosphate) ---------------------------- 17.5 ul
Color Development Buffer ----------------------- 5 ml




PROCEDURES

Preparation of Digoxigenin-Labeled Probe

1. Label the probe DNA with digoxigenin.

2. Prepare DIG-labeled probe cocktail as follow.

100 x Denhardt's Solution ---------------------------- 5 ul
50% Dextran sulfate ---------------------------------- 50 ul
Salmon sperm DNA (10 mg/ml) ---------------------- 10 ul
20 x SSC -------------------------------------------- 100 ul
Digoxigenin-labeled probe ---------------------------- 500 ng
Deionized Formamide -------------------------------- 250 ul
Add distilled H2O to make a final volume of --------- 500 ul

  • Store at -20oC.


    Preparation of Tissue Cross Section

    3. Wash slides overnight in a detergent.

    4. Next morning, wash slides in tap water.

    5. Rinse slides in deionized water.

    6. Air-dry the slides.

    7. Wash slides in acetone for 5-10 min.

    8. Incubate slides in a Silicone Solution for 10 min.

    9. Rinse the slides in distilled water.

    10. Air-dry the slides.

    11. Fix tissues in Formaldehyde Fixatives for 4-6 hours.

    12. Embed tissue in paraffin as suggested.

    13. Make 10 um sections of tissue.

    14. Place cross-sections on the pretreated slides.

    15. Bake the sections on the slides for 30 min at 60oC.

    16. Dewax the sections on the slides in xylene for 30 min. Repeat two times with new xylene.

    17. Rehydrate them in a graded ethanol series as follow.

    Step
    Ethanol
    Time
    1
    100%
    5 min
    2
    100%
    5 min
    3
    95%
    5 min
    4
    95%
    5 min
    5
    70%
    5 min
    6
    H2O
    5 min


    18. Treat each section with 30 ul of Proteinase K Solution for 15 min at 37oC.
  • During the proteolytic digestion cover the sections with siliconized coverslips and place them in a humid chamber.


    Hybridization and Signal Detection

    19. Remove the coverslips and fix the sections in 0.4% Formaldehyde for 5 min at 4oC.

    20. Immerse the sections in distilled H2O for 5 min.

    21. Allow the sections to drip and air dry for 5 min.

    22. Distribute about 5-20 ul of the probe cocktail over each section.
  • Prepare a negative control by covering one section from each block with a "blind" probe cocktail, i.e. a probe cocktail containing all the ingredients except the labeled probe DNA.

    23. Place siliconized coverslips over each section and denature the DNA by placing the slides on a heating plate at 95oC for 5 min.

    24. Cool the slides for 1 min on ice.

    25. Place the slides in a humid chamber and incubate for 3 hours to overnight at 42oC in an oven.

    26. Remove the coverslips and wash the sections as follow.

    Step
    Solution
    Temperature
    Time
    1
    2 x SSC
    20oC
    5 min
    2
    2 x SSC
    20oC
    5 min
    3
    0.1 x SSC
    42oC
    10 min
    4
    0.1 x SSC
    42oC
    10 min


    27. Dip each section on the slides into Pretreatment Buffer.

    28. Cover each section with 20-40 ul Blocking Buffer.

    29. Put a coverslip.

    30. Incubate sections at 20oC for 15 min.

    31. Remove the coverslips and dip the sections in Pretreatment Buffer.

    32. Dilute the antibody conjugate 1:500 in Blocking Buffer.

    33. Distribute 10-20 ul of the diluted conjugate over each section, including the negative controls.

    34. Place a coverslip over each section and place all sections in a humid chamber at room temperature for 1 hour.

    35. Remove the coverslips.

    36. Wash the sections 10 min in Pretreatment Buffer. Repeat two times.

    37. Equilibrate the sections for 5 min in Color Development Buffer.

    38. Distribute 20 ul Color Substrate Solution onto each section.
  • Cover with a coverslip and leave in the dark for 1 hour to overnight.

    39. After the incubation, remove the coverslips.

    40. Wash the sections gently with distilled water.

    41. Stain a few seconds with neutral red and mount.




    NOTES

  • The coated slides can be stored dry under dust-free conditions for several months.

  • The proteolytic treatment is crucial and may need to be varied with different tissues.
    When too much Proteinase K is used, the tissue totally disintegrates, and if too little is used positive signals may be totally absent.




  • KIT INFORMATION




    REFERENCES



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