Molecular Techniques and Methods

In situ Hybridization
Detection of mRNA on Paraffin Embedded Tissue

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Fixatives (300 ml)
4% Paraformaldehyde ------------------------------ 12 g
(or, 2.5% Glutaraldehyde --------------------------- 30 ml of 25% Glutaraldehyde)
100 mM Sodium Phosphate Buffer ----------------- 30 ml of 1 M Na-Phosphate Buffer
Add distilled H2O to make a final volume of ------- 300 ml
  • Stir overnight. Paraformaldehyde slowly dissolves in aqueous solution.
  • Filter by 0.2 um membrane before use.


    Silicone Solution (100 ml)
    2% 3-Aminopropyltriethoxysilane ------------------ 2 ml
    Acetone ------------------------------------------- 98 ml


    TBS Buffer (1 liter)
    50 mM Tris-HCl (pH 7.5) ------------------------- 50 ml of 1 M Tris-HCl
    150 mM NaCl ------------------------------------- 30 ml of 5 M NaCl
    Distilled H2O --------------------------------------- 920 ml


    Tris-Acetic Anhydride Solution (300 ml)
    0.5% Acetic anhydride ----------------------------- 1.5 ml
    100 mM Tris-HCl (pH 8.0) ------------------------ 30 ml of 1 M Tris-HCl
    Distilled H2O --------------------------------------- 300 ml


    Proteinase K Solution (300 ml)
    Proteinase K ----------------------------------------- 3-50 mg
    2 mM CaCl2 ----------------------------------------- 600 ul of 1 M CaCl2
    TBS -------------------------------------------------- 300 ml


    Hybridization Buffer (10 ml)
    2 x SSC ---------------------------------------------- 1 ml of 20 x SSC
    10% Dextran sulfate ---------------------------------- 1 g
    Sheared Salmon Sperm DNA (1 ug/ml) --------------- 1 ul of Salmon Sperm DNA (10 mg/ml)
    0.02% SDS ------------------------------------------ 20 ul of 10% SDS
    50% Deionized Formamide --------------------------- 5 ml of 100% Deionized Formamide
    Add distilled H2O to make a final volume of --------- 10 ml


    Blocking Solution (200 ml)
    150 mM NaCl ---------------------------------------- 6 ml of 5 M NaCl
    100 mM Tris-HCl (pH 7.5) --------------------------- 20 ml of 1 M Tris-HCl (pH 7.5)
    0.5% BSA (Bovine Serum Albumin) ------------------ 1 g
    0.5% Blocking Reagent ------------------------------- 10 ml of 10% Blocking Reagent
    Distilled H2O ----------------------------------------- 164 ml


    Washing Solution 1 (200 ml)
    50% Deionized Formamide --------------------------- 100 ml of 100% Deionized Formamide
    1 x SSC ---------------------------------------------- 10 ml of 20 x SSC
    Distilled H2O ----------------------------------------- 9 ml


    Washing Solution 2 (200 ml)
    1 x SSC ---------------------------------------------- 10 ml of 20 x SSC
    Distilled H2O ----------------------------------------- 190 ml


    Color Development Buffer (200 ml)
    100 mM Tris-HCl ------------------------------------ 20 ml of 1 M Tris-HCl
    100 mM NaCl (pH 7.5) ------------------------------ 4 ml of 5 M NaCl
    50 mM MgCl2 --------------------------------------- 10 ml of 1 M MgCl2
    Distilled H2O ----------------------------------------- 166 ml


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




    PROCEDURES

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


    Tissue Fixation and Sectioning

    1. Fix tissues for 3-4 hours at room temperature in Fixatives.

    2. After fixation, wash the tissues in PBS.

    3. Embed the fixed tissues in paraffin.

    4. Coat slides with a Silicone Solution.
  • Air dry them.

    5. Cut 10 um thick sections from the fixed tissue and attach them to the prepared slides.

    6. Dry the sections overnight at 55oC.

    7. Dewax slides extensively by treating with 3 changes of xylene overnight.

    8. To preserve the mRNA during the following procedure, fix the sections once again with Fixatives for 20 min.

    9. Rinse the sections 5 times with TBS Buffer.

    10. Treat the sections for 10 min with 200 mM HCl to denature proteins.

    11. Rinse the sections 5 times with TBS Buffer.

    12. Incubate the sections for 10 min on a magnetic stirrer with a freshly mixed Tris-Acetic Anhydride Solution.

    13. Rinse the sections 5 times with TBS Buffer.

    14. Treat the sections for 20 min at 37oC with Proteinase K Solution.

    15. Rinse the sections 3-5 times with TBS Buffer.

    16. Incubate the sections at 4oC for 5 min with TBS Buffer (pH 7.5) to stop the Proteinase K digestion.

    17. Dehydrate the sections in a graded series of ethanol solutions.

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


    18. Rinse the sections briefly with chloroform.


    Hybridization and Signal Detection

    19. Place the sections in a humid chamber at 55oC for 30 min.

    20. Dilute the labeled antisense RNA probe in Hybridization Buffer.
  • The diluted probe solution may contain as much as 1 part labeled probe to 4 parts Hybridization Buffer.

    21. Pipette the diluted antisense RNA probe solution onto each section at a volume of 10 ul/cm. Cover with a coverslip.

  • A prehybridization step with Hybridization Buffer alone does not improve results.
  • As a control serve sections treated in the same way whereby a labeled sense RNA probe is used.
  • Control hybridization with sense RNA probes is necessary to prove the specificity of the reaction.

    22. Place the slides on a hot plate at 95oC for 5 min.
  • This step increases the signal from RNA/RNA hybrids.

    23. Incubate the slides in a humid chamber for 4-6 hours at 50-75oC.
  • Hybridization specificity can be increased by increasing the temperature of hybridization, if necessary, as high as 75oC.
  • Increasing the temperature can help to differentiate mRNAs of highly homologous proteins.

    24. Incubate the slides in 2 x SSC overnight.
  • The coverslips will float off during this incubation.

    25. Wash the slides as follows.

    Step
    Solution
    Temperature
    Time
    1
    Washing Solution 1
    55oC
    20 min
    2
    Washing Solution 1
    55oC
    20 min
    3
    Washing Solution 1
    55oC
    20 min
    4
    Washing Solution 2
    23oC
    15 min
    5
    Washing Solution 2
    23oC
    15 min


    26. Rinse the sections 3-5 times with TBS Buffer.

    27. Incubate the slides for 15 min with Blocking Solution.

    28. Incubate the slides for 60 min with Alkaline-Phosphatase-conjugated anti-DIG Antibody diluted 1:500 in Blocking Solution.

    29. Rinse the sections 3-5 times with TBS Buffer.

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

    38. Distribute 20 ul Color Substrate Solution onto each section.

    39. Cover with a coverslip and leave in a Coplin jar in the refrigerator, incubate the sections with Color Substrate Solution for 1-24 hours or until sufficient color develops.

  • Incubation can be extended up to 120 hours if the Color Substrate Solution is replaced every time it precipitates or changes color.

    40. Stop the color reaction by rinsing the slides several times with tap water.

    41. Finally, rinse the slides with distilled water.

    42. Mount the slides directly with any water-soluble mounting medium.




    NOTES

  • The concentration of Proteinase K depends upon the degree of fixation. Generally, start with 20 ug/ml Proteinase K for paraformaldehyde-fixed tissue. The concentration needed for glutaraldehyde-fixed or overfixed tissue is generally higher than for paraformaldehyde-fixed tissue. Determine the optimal concentration of Proteinase K empirically for each type of fixation. One of the most critical steps in the whole procedure is finding the balance between prior fixation and the proper concentration of Proteinase K. Proteinase K concentrations which are too low or too high may lead to false negative results. Autolysis causes fewer problems than overfixation does. In overfixed material, we were not always successful in demonstrating mRNA.

  • Do not postfix the sections with paraformaldebyde after Proteinase K digestion, since this reduces signal intensity.

  • The concentration of dextran sulfate is critical. Without appropriate amounts of dextran sulfate, the method loses sensitivity. However, excessive concentrations of dextran sulfate causes higher viscosity of the Hybridization Buffer. To prevent uneven distribution of the probe and uneven signals, always vortex the Hybridization Buffer extensively.

  • The amount of probe needed depends upon the degree of probe labeling. Generally, use the lowest probe concentration that gives optimal response in that dot blot procedure.


  • KIT INFORMATION


    REFERENCES

  • McKhann HI and Hirsch AM. 1993. In situ localization of specific mRNAs in plant tissues. In "Methods in Plant Molecular Biology and Biotechnology". Glick BR and Thompson JE eds. pp179-205. CRC press. Boca Raton, Ann Arbor, London, Tokyo.


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

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