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In Situ Hybridization Using S35 Labeling of RNA Probe (Paraffin Sections)

Sectioning

Cut 6-10 micron sections under RNase free conditions. Using RNase free water for floating the sections. Wear gloves when sectioning. Fisher brand Superfrost/Plus slides can be used for picking sections. Store slides at 4 °C. (Slides can be used for one year. )

Dewaxing and Pretreatment of Slides

  1. Warm slides at 55-60 °C for 1-1.5 hour.
  2. Deparaffinize for 20min X 2 with 55-60 °C xylene.
  3. Rehydrate:
    100% ETOH 2min X2
    95% ETOH 2min
  4. Wash with 0.9% NaCl for 5min.
    5M NaCl 31.5mL to 1000ml DepH2O
  5. Incubate with 0.2M HCl for 15min at RT.
    3.4mL 12N HCl to 200ml H2O
  6. PBS 1min X 3.
  7. Incubate with Proteinase K 20ug/mL in 50mM Tris pH 7.6, 5mM
    EDTA for 8min at RT.
    1M Tris 7.6 10ml
    0.5 M EDTA 2ml
    ddH2O 188mL
    PK (10mg/ml) 400uL (aliquot in -20°C, to be added immediately before incubation.)
  8. 0.4% Glycine in PBS for 2min X 2 at RT.
    Glycine 1.8g to 450ml PBS
  9. 0.9% NaCl for 2min X 2.
  10. 0.1M Triethanolamine-HCl pH 8.0 on magnetic stirrer.

    Stock: 1M Triethanolamine pH 8.0 (1000ml)
     Triethanolamine: 185.7g
    10N NaOH (20g NaOH in 50ml DepH2O) 46-47ml
    Add DepH2O to total volume 1000ml. Check pH

    Working Solution:
    0.1M Triethanolamine pH 8.0: (200ml)
     1M Triethanolamine 20mL
    ddH2O 180mL

    Place RNAse free stir bar into staining dish. Place dish on stirrers.

  11. Add 500uL of Acetic Anhydride to staining dish while stirring.
    Stir for 5min. Add again 500uL of Acetic Anhydride. Stir for 5min.
  12. Rinse in Dep H2O X2.
  13. Dehydration:
    95% EtOH 1min
    100% EtOH 1minX2
  14. Air dry in dust free area. Slides can then proceed to hybridization step or be stored at -80 °C.

Hybridization

Hyb. Mix:

Final Concentration Stock 30mL

50% Formamide 100% 15mL

20mM Tris pH8.0 1M 600uL

0.3M NaCl 5M 1.8mL

5mM EDTA 0.5M 300uL

10% Dextran Sulfate 50% 6mL

Denhardts 50X 600uL

0.5mg/mL tRNA 10mg/mL 1.5mL

10mM DTT 1M 300uL

DEP H2O 3.9mL

Hyb. Mix is aliquoted and stored at -80 °C.

  1. Thaw Hyb. Mix .
  2. Add Hyb. Mix to each new tube (90uL per slide)
  3. Add probe to make final concentration at 70,000cpm/ul to each tube.
    70,000cpm/uL X 90ul= 6.3 X 106cpm
    Example:
    819,966cpm/2uL = 409,998/ul (probe concentration)
    6,300,000/409,983 = 15.4ul/slide
  4. Mix probe by vortex and then spin down probe, keep mixed probe on ice.
  5. Boil each probe for 2min.
  6. Immediately add 80ul probe to 24 X 50mm coverglass. Turn slides tissue side down and contact tissue to the probe. Turn slides back with coverglass at top.
  7. Hybridize overnight at 55 °C in a humidified chamber ( 50% formamide and 2X SSC).

Posthybridization:

  1. Warm 2X SSC at 60°C and STE at 37 °C.
  2. Put slides into washing container with prewarmed 2XSSC.
  3. Remove coverglass from slides. Chek no coverglass left in slides rack. Wash for 15min at 60°C.
  4. Change prewarmed 2X SSC and wash for 30min at 60°C.
  5. Wash with STE at 37°C for 20min.
    1XSTE: (1000ml)
    20X SSC 200ml
    1M Tris pH7.6 20ml
    0.5M EDTA 2ml
    ddH2O 778ml
  6. RNase treatment for 45 min at 37°C. (6-20ug/ml in STE)
    RNase treatment:
    1XSTE 200mL 350 ml
    RNase(10mg/ml) 250uL 400ul
  7. Wash with STE at 37°C for 10min.
  8. Wash with 2X SSC for 30 min at 60°C.
  9. Wash with 0.5X SSC for 30 min at 60°C.
  10. Wash with 0.1X SSC for 30 min at 60°C.
  11. Rinse with 0.1X SSC at RT.
  12. ddH2O at RT X2.
  13. Rinse with 95% ETOH.
  14. Air dry in dust free area.
  15. Dried slides are exposed to a BioMax Kodak film for 1 to 3 days.

Dipping

* For working with emulsion, use a dark red Kodak safelight No 2.

* Aliquot emulsion: Dilute 118ml Kodak NTB-2 emulsion with 200ml ddH2O (prewarmed at at 42-45 °C). Warm emulsion at 42-45 °C until completely melted. ( about 12min) . Aliquot 20ml to glass scintillation vials or 50ml tubes. Wrapped in two layers of aluminum foil and stored in dark at 4 °C. Check the background of emulsion by dipping blank slides in one aliquot. Keep slides in dark at room temperature for 4-6 hours. Develop slides when slides were dry.

  1. Melt emulsion by warm the emulsion vial at 42-45 °C water bath for about 12 min. Pour emulsion into special slides coating jar bathed in 42-45 °C.
    (Avoid keeping emulsion at 45 °C for longer than 40 min.)
  2. Dip slides for 4 seconds, then drain for 3 seconds.
  3. Place slides horizontally on a rack at RT in the dark for 6 hours or overnight. ( Room should be cool )
  4. Slides are placed in black slides boxes. Wrapped with aluminum foil and stored in dark at 4 °C.
  5. Exposure one week . ( 3days to 2 weeks )

Developing and Staining

* Prepare a 10mg/ml stock solution of Hoechst 33258 (Sigma, bis- benzimide) in DMSO. Make 50ul aliquots and store at -80°C.

* Make Kodak D-19 developer and Kodak fixer solutions according to the instructions on the package. The solutions should be made one day earlybefore to use. Both solutions shelf life are two months.

  1. Equilibrate slides to RT for 1 hour.
  2. Open slides box in dark and transfer it into a slides rack.
  3. Slides are developed in Kodak D-19 developer for 2 min at RT (no longer, no movement ).
  4. Rinse in tap water for 30 seconds at RT ( no movement ).
  5. Immerse in Kodak fixer for 5 min at RT ( no movement ).
  6. Rinse in ddH2O for 10 min ( 2 or 3 changes, no movement ).
  7. Dilute Hoechst dye stock solution in H2O to make a final concentration of 2ug/ml.
    H2O 250ml
    Hoechst stock 50ul
  8. Developed slides are placed into the dye solution for 2min.
  9. Rinse 2 min in stationary water ( no longer ).
  10. Rinse one or more under running deionized water.
  11. Use a new and sharp razor blade to remove the emulsion at backside of the slides.
  12. Rinse with deionized water.
  13. Shake off excess water and air dry slides at RT.
  14. Cover sections with one drop of 5g Canada Balsam/10ml methyl salicylate, lay on a coverslip. Keep slides horizontal until edges harden.

Diethyl pyrocarbonate treated H2O (DEP H2O):
0.1% Diethyl pyrocarbonate in ddH2O. Shake well and make sure the Diethyl pyrocarbonate completely dissolved. Incubate at 37 °C overnight. Autoclave 15 min.

Glassware treatment:
Glassware and stir bar can be baked at 180-220 °C overnight to inhibit RNase.

 

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