Molecular Techniques and Methods

Whole Mount In situ Hybridization (WISH)

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The procedure for in situ hybridization to mRNA on cross-sections is rather tedious and time-consuming. It depends on the quality of the cross-sections and particularly on the quality of the probe. In contrast, analysis of the mRNA in whole tissue simplified the procedure and allowed a much higher resolution.




MATERIALS AND SOLUTIONS

Formaldehyde Fixation Buffer (10 ml)
5% Formaldehyde ------------------------------------- 1.35 ml of 37% Formaldehyde
0.1 M HEPES (pH 6.9) -------------------------------- 1 ml of 1 M HEPES
2 mM MgSO4 ----------------------------------------- 20 ul of 1 M MgSO4
1 mM EGTA (pH 8.0) --------------------------------- 20 ul of 0.5 M EGTA
1 mM Levamisole -------------------------------------- 2.4 mg
Add DEPC-treated H2O to make a final volume of --- 10 ml
  • Adjust pH 6.9.


    Paraformaldehyde Fixation Buffer (10 ml)
    4% Paraformaldehyde ---------------------------------- 0.4 g
    0.1 M HEPES (pH 6.9) -------------------------------- 1 ml of 1 M HEPES
    2 mM MgSO4 ----------------------------------------- 20 ul of 1 M MgSO4
    1 mM EGTA (pH 8.0) --------------------------------- 20 ul of 0.5 M EGTA
    1 mM Levamisole -------------------------------------- 2.4 mg
    Add DEPC-treated H2O to make a final volume of --- 10 ml
  • Adjust pH 6.9.


    PBT (100 ml)
    PBS ---------------------------------------------------- 99 ml
    0.1% Tween-20 ---------------------------------------- 1 ml of 10% Tween-20
    1 mM Levamisole -------------------------------------- 24 mg


    ME Solution (100 ml)
    Methanol ---- ------------------------------------------ 90 ml
    0.05 M EGTA ----------------------------------------- 10 ml of 0.5 M EGTA
    1 mM Levamisole -------------------------------------- 24 mg


    PP Solution (100 ml)
    4% Paraformaldehyde ---------------------------------- 0.4 g
    PBS --------------------------------------------------- 100 ml
    1 mM Levamisole -------------------------------------- 24 mg


    Proteinase K Solution (10 ml)
    Proteinase K ------------------------------------------- 250 ul of Protinase K (20 mg/ml)
    PBS ---------------------------------------------------- 9.75 ml


    Hybridization Solution (10 ml)
    50% Deionized Formamide ----------------------------- 5 ml of 100% Deionized Formamide
    5 x SSC ------------------------------------------------ 2.5 ml of 20 x SSC
    Type II-C Torula yeast RNA --------------------------- 1 ml of Type II-C Torula yeast RNA (10 mg/ml)
    1 x Denhardt's ------------------------------------------ 0.1 ml of 100 x Denhradt's
    5 mM EDTA ------------------------------------------- 0.1 ml of 0.5 M EDTA
    0.1% Tween-20 ---------------------------------------- 0.1 ml of 10% Tween-20
    Salmon Sperm DNA ----------------------------------- 1 ml of Salmon Sperm DNA (10 mg/ml)
    1 mM Levamisole -------------------------------------- 2.4 mg


    Color Development Buffer (10 ml)
    100 mM NaCl ----------------------------------------- 0.2 ml of 5 M NaCl
    50 mM MgCl2 ----------------------------------------- 0.5 ml of 1 M MgCl2
    100 mM Tris-HCl (pH 9.5) ---------------------------- 1 ml of 1 M Tris-HCl
    1 mM Levamisole -------------------------------------- 2.4 mg
    0.1% Tween-20 ---------------------------------------- 1 ml of 10% Tween-20
    Distilled H2O ------------------------------------------- 7.3 ml
  • Levamisole is a potent inhibitor of endogenous Lysosomal Phosphatases.


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




    PROCEDURES

    Preparation and Fixation of Tissue

    1. Wash tissue sample with distilled H2O.

    2. Wash tissue sample with 0.1 % Triton X-100.

    3. Fix the tissue sample either with (a) Formaldehyde or (b) Paraformaldehyde Fixation Buffer.


    (a) Formaldehyde Fixation

    4. Transfer tissue sample into a glass scintillation vial containing 4 ml of Formaldehyde Fixation Buffer.

    5. Shake the vial for 15-20 min.

    6. Remove the Fixation Buffer and add 10 ml Methanol.

    7. If tissue sample sink to the bottom at this stage, store at -20oC.

  • If tissue sample do not sink to the bottom at this stage, remove and add new methanol.

    8. Continue on step 8 in tissue preparation.



    (b) Paraformaldehyde Fixation

  • This protocol is more laborious, but leads to a lower background and to better preservation of the morphology.

    4. Transfer tissue sample into a glass scintillation vial containing 4 ml of Paraformaldehyde Fixation Buffer.

    5. Shake the vial for 15-20 min.

    6. Remove the Fixation Buffer and add 10 ml Methanol.

    7. If tissue sample sink to the bottom at this stage, store at -20oC.

  • If tissue sample do not sink to the bottom at this stage, remove and add new methanol.



    Tissue Preparation

    8. Transfer each tissue sample to a tube containing ME Solution.

    9. Refix the tissue sample and dehydrate by passage through the following series of solutions containing ME Solution and PP Solution.

    Step
    ME Solution
    PP Solution
    Time
    1
    70 ml
    30 ml
    5 min
    2
    50 ml
    50 ml
    5 min
    3
    30 ml
    70 ml
    5 min
    4
    -----
    100 ml
    20 min


    10. Wash the tissue sample in PBS for 10 min.



    Pretreatment of Fixed Tissue Samples

  • Perform all steps under RNase-free conditions!
  • Prepare all solutions and buffers with DEPC-treated.
  • Bake all glassware for 4 hours at 180oC.


    11. Place each fixed tissue sample in a 1.5 ml microcentrifuge tube.

    12. Wash each tissue sample in 1 ml PBT for 5 min. Repeat 3 times.
  • Tween-20 reduces non-specific adhesion of the tissue samples to the plastic surfaces.

    13. Incubate the tissue sample for 3-5 min in 1 ml of Proteinase K Solution.

    14. Stop the digestion by removing the Proteinase K solution.

  • The Proteinase K digestion time is critical.
  • If it is too short, the background increases and sensitivity is lost.
  • If it is too long, the tissue burst during the subsequent steps.

    15. Add 1 ml PBT containing 2 mg/ml Glycine to the tissue sample.

    16. Incubate for 2 min.

    17. After the Proteinase K digestion, wash the tissue sample in 1 ml PBT for 5 min. Repeat.

    18. Refix tissue sample for 20 min in 1 ml PP Solution.

    19. Wash tissue sample in 1 ml PBT for 10 min. Repeat 3 times.

    20. Prepare digoxigenin-labeled probes.



    Pre-hybridization and Signal Detection

    21. Pre-hybridize the tissue samples as follows.

    Step
    Hybridization
    Solution
    PBT
    Temperature
    Time
    1
    5 ml
    5 ml
    23oC
    1 hr
    2
    10 ml
    --------
    23oC
    1 hr
    3
    10 ml
    --------
    45oC
    1-24 hr


    22. Heat denature the probe in the presence of 10 ug Salmon Sperm DNA.
  • The Salmon Sperm DNA should effectively compete out simple sequences and thus reduce the background.

    23. Dilute the probe to approximately 0.5 ug labeled probe per ml in Hybridization Solution.

    24. Remove most of the prehybridization supernatant.

    25. Add the heat-denatured Probe in Hybridization Solution.

    26. Hybridize overnight at 45oC in a water bath.

    27. Wash the tissue samples at room temperature in each of the following solutions.

    Step
    Hybridization
    Solution
    PBT
    Time
    1
    10 ml
    -------
    20 min
    2
    8 ml
    2 ml
    20 min
    3
    6 ml
    4 ml
    20 min
    4
    4 ml
    6 ml
    20 min
    5
    2 ml
    8 ml
    20 min
    6
    --------
    10 ml
    20 min
    7
    --------
    10 ml
    20 min

  • Use a less extensive washing protocol if probe produces a low background.


    28. Prepare a fresh dilution (1: 2,000 to 1: 5,000) of alkaline phosphatase-conjugated anti-DIG-antibody in PBT.

    29. Incubate each tissue sample with 500 ul freshly diluted antibody conjugate for 1 hour at room temperature on a revolving wheel.

    30. Wash the tissue sample in PBT for 20 min. Repeat 4 times.

    31. Wash the tissue sample in Color Development Buffer for 5 min. Repeat 3 times.

    32. Bring the tissue sample into a small dish with 1 ml of Color Substrate Solution.

    33. Develop the color for 10 min to overnight in the dark.
  • Color development may occasionally be controlled under the binocular microscope and stopped in PBT before background develops.

    34. Dehydrate the tissue sample in a series of ethanol solutions and mount.

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




    NOTES



    KIT INFORMATION



    REFERENCES



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

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