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HIDEFUMI YOSHIOKA AND SUMIHARE NOn
Deparaffinization and pretreatment
1. Deparaffinize sections(20 slides in one carrier) by dipping sequentiallyfor 5 mins each in: xylene three times, 100% ethanol
three times, 90% ethanol, 80% ethanol, 70% ethanol, 50%
ethanol, and ISHRI for 10 mins.
2. Immerse in ISHRI containing ISHR6 (final concentration:
1 - 10 )lg/ml depending on samples)
3. Quench the proteinase activity in ISHR2 for 10 mins.
4. Wash for 3 mins in ISHRI twice.
5. Acetylate the sections in ISHR3 by adding 1 ml of ISHR4
slowly with vigorous agitation for 15 mins.
6. Wash the slides for 10 mins in ISHR5 twice.
7. Prehybridize in 200 ml of a prehybridization solution of 50%
formamide/ISHR5 containing 10 mM DTT for 30 mins at
50° C.
Hybridization 1. Prepare hybridization mixture by adding RNA probe (5x10 4
dpml)ll) in ISHR7.
2. Aspirate excess prehybridization mixture with a capillary
tube.
3. Prepare sheets of Parafilm (2.5 cm x3.0 cm) for cover.
4. Spot 20 )ll of the mixture on each slide.
5. Cover the sections with a sheet of Parafilm.
6. Place four slides on two glass rods in a rectangular plate.
7. Add prehybridization solution to humidify inside of the plate.
8. Seal the plate with vinyl tape.
9. Incubate at 50° C at least for 16 hrs.
Washing 1. Immerse the slides in 50% formamide/2xSSC containing
10 mM DTT (Use the prehybridization mixture).
2. Wait until the Parafilm floats off.
3. Transfer the slides to a slide carrier (use only for washing).
HIDEFUMI YOSHIOKA AND SUMIHARE NOn
Deparaffinization and pretreatment
1. Deparaffinize sections(20 slides in one carrier) by dipping sequentiallyfor 5 mins each in: xylene three times, 100% ethanol
three times, 90% ethanol, 80% ethanol, 70% ethanol, 50%
ethanol, and ISHRI for 10 mins.
2. Immerse in ISHRI containing ISHR6 (final concentration:
1 - 10 )lg/ml depending on samples)
3. Quench the proteinase activity in ISHR2 for 10 mins.
4. Wash for 3 mins in ISHRI twice.
5. Acetylate the sections in ISHR3 by adding 1 ml of ISHR4
slowly with vigorous agitation for 15 mins.
6. Wash the slides for 10 mins in ISHR5 twice.
7. Prehybridize in 200 ml of a prehybridization solution of 50%
formamide/ISHR5 containing 10 mM DTT for 30 mins at
50° C.
Hybridization 1. Prepare hybridization mixture by adding RNA probe (5x10 4
dpml)ll) in ISHR7.
2. Aspirate excess prehybridization mixture with a capillary
tube.
3. Prepare sheets of Parafilm (2.5 cm x3.0 cm) for cover.
4. Spot 20 )ll of the mixture on each slide.
5. Cover the sections with a sheet of Parafilm.
6. Place four slides on two glass rods in a rectangular plate.
7. Add prehybridization solution to humidify inside of the plate.
8. Seal the plate with vinyl tape.
9. Incubate at 50° C at least for 16 hrs.
Washing 1. Immerse the slides in 50% formamide/2xSSC containing
10 mM DTT (Use the prehybridization mixture).
2. Wait until the Parafilm floats off.
3. Transfer the slides to a slide carrier (use only for washing).