In Situ Hybridization Using S35 Labeling of RNA Probe (Paraffin Sections)
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
- Warm slides at 55-60 °C for 1-1.5 hour.
- Deparaffinize for 20min X 2 with 55-60 °C xylene.
100% ETOH 2min X2
95% ETOH 2min
- Wash with 0.9% NaCl for 5min.
5M NaCl 31.5mL to 1000ml DepH2O
- Incubate with 0.2M HCl for 15min at RT.
3.4mL 12N HCl to 200ml H2O
- PBS 1min X 3.
- 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
PK (10mg/ml) 400uL (aliquot in -20°C, to be added immediately before incubation.)
- 0.4% Glycine in PBS for 2min X 2 at RT.
Glycine 1.8g to 450ml PBS
- 0.9% NaCl for 2min X 2.
- 0.1M Triethanolamine-HCl pH 8.0 on magnetic stirrer.
Stock: 1M Triethanolamine pH 8.0 (1000ml)
10N NaOH (20g NaOH in 50ml DepH2O) 46-47ml
Add DepH2O to total volume 1000ml. Check pH
0.1M Triethanolamine pH 8.0: (200ml)
1M Triethanolamine 20mL
Place RNAse free stir bar into staining dish. Place dish on stirrers.
- Add 500uL of Acetic Anhydride to staining dish while stirring.
Stir for 5min. Add again 500uL of Acetic Anhydride. Stir for 5min.
- Rinse in Dep H2O X2.
95% EtOH 1min
100% EtOH 1minX2
- Air dry in dust free area. Slides can then proceed to hybridization step or be stored at -80 °C.
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.
- Thaw Hyb. Mix .
- Add Hyb. Mix to each new tube (90uL per slide)
- Add probe to make final concentration at 70,000cpm/ul to each tube.
70,000cpm/uL X 90ul= 6.3 X 106cpm
819,966cpm/2uL = 409,998/ul (probe concentration)
6,300,000/409,983 = 15.4ul/slide
- Mix probe by vortex and then spin down probe, keep mixed probe on ice.
- Boil each probe for 2min.
- 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.
- Hybridize overnight at 55 °C in a humidified chamber ( 50% formamide and 2X SSC).
- Warm 2X SSC at 60°C and STE at 37 °C.
- Put slides into washing container with prewarmed 2XSSC.
- Remove coverglass from slides. Chek no coverglass left in slides rack. Wash for 15min at 60°C.
- Change prewarmed 2X SSC and wash for 30min at 60°C.
- Wash with STE at 37°C for 20min.
20X SSC 200ml
1M Tris pH7.6 20ml
0.5M EDTA 2ml
- RNase treatment for 45 min at 37°C. (6-20ug/ml in STE)
1XSTE 200mL 350 ml
RNase(10mg/ml) 250uL 400ul
- Wash with STE at 37°C for 10min.
- Wash with 2X SSC for 30 min at 60°C.
- Wash with 0.5X SSC for 30 min at 60°C.
- Wash with 0.1X SSC for 30 min at 60°C.
- Rinse with 0.1X SSC at RT.
- ddH2O at RT X2.
- Rinse with 95% ETOH.
- Air dry in dust free area.
- Dried slides are exposed to a BioMax Kodak film for 1 to 3 days.
* 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.
- 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.)
- Dip slides for 4 seconds, then drain for 3 seconds.
- Place slides horizontally on a rack at RT in the dark for 6 hours or overnight. ( Room should be cool )
- Slides are placed in black slides boxes. Wrapped with aluminum foil and stored in dark at 4 °C.
- 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.
- Equilibrate slides to RT for 1 hour.
- Open slides box in dark and transfer it into a slides rack.
- Slides are developed in Kodak D-19 developer for 2 min at RT (no longer, no movement ).
- Rinse in tap water for 30 seconds at RT ( no movement ).
- Immerse in Kodak fixer for 5 min at RT ( no movement ).
- Rinse in ddH2O for 10 min ( 2 or 3 changes, no movement ).
- Dilute Hoechst dye stock solution in H2O to make a final concentration of 2ug/ml.
Hoechst stock 50ul
- Developed slides are placed into the dye solution for 2min.
- Rinse 2 min in stationary water ( no longer ).
- Rinse one or more under running deionized water.
- Use a new and sharp razor blade to remove the emulsion at backside of the slides.
- Rinse with deionized water.
- Shake off excess water and air dry slides at RT.
- 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 and stir bar can be baked at 180-220 °C overnight to inhibit RNase.