118
HIDEFUMI YOSHIOKA AND SUMIHARE NOJI
- ISHR4: Two 1 ml Acetic anhydride
- ISHR5: two 1 I solutions containing 4xSSC containing 0.6
M NaCI, 0.06 M sodium citrate.
- ISHR6: Proteinase K solution (10 mg/ml) 40 fll
- ISHR7: Eight 93 fll hybridization buffers containing
- 50% formamide, 2xSSC, 1 flglfll tRNA, 1 flglfll sonicated
salmon DNA,
- 1 flglfll bovine serum albumins (BSA), and 10% dextran
sulfate.
- ISHR8: 100 fll dithiothreitol (DTT) solution (1.2 M)
- ISHR9: 800 ml NTE buffer containing 0.5M NaCl, 10 mM
Tris-HCI (pH 8.0), and 1 mM ethylene diaminse tetra acetate sodium salt (2NaEDTA)
- ISHRlO: Two 0.4 ml solutions containing RN ase A solution
(10 mg/ml RNase A).
- ISHR11: Two 600 ml solutions containing O.lxSSC, 15 mM
NaCI and 15mM sodium.
Equipment Disposable gloves, two forceps, two standard slide glass carriers
and supplies (20 slots), 29 rectangular staining dishes, 500 ml beaker, a starto be pre- ring bar, stirrer, clamp, pipettes, 200 ml and 1 I measuring cypared linder, Parafilm, incubator, rectangular culture dishes, 8 glass
rods, 1 ml micropipettes, and yellow tips
Reagents Fixative (50 m14% paraformaldehyde):
- 10 ml of 20% paraformaldehyde (use within a month). (To
prepare 20% paraformaldehyde, warm water and paraformaldehyde powder at 50-60°C and add ION NaOH until solution
becomes clear.)
- 40 ml of PB (final 0.07-0.08 M)
- PB: solution A: 0.2 M NaH2P0 4 • H 2 0 (27.6 gil)
- solution B: 0.2 M NaH2P04 • 2 H20 (35.6 gil)
- 23 ml solution A + 77 ml solution B + 100 ml H20 = 200 ml
(0.1 M)
- PBS: 5.84 g NaCl + 1.44 g NaH2P04 • 2 H20 + 0.20 g NaH2P04
in 1000 ml water, 450 ml formamide, 920 ml ethanol, 200 ml
xylene
HIDEFUMI YOSHIOKA AND SUMIHARE NOJI
- ISHR4: Two 1 ml Acetic anhydride
- ISHR5: two 1 I solutions containing 4xSSC containing 0.6
M NaCI, 0.06 M sodium citrate.
- ISHR6: Proteinase K solution (10 mg/ml) 40 fll
- ISHR7: Eight 93 fll hybridization buffers containing
- 50% formamide, 2xSSC, 1 flglfll tRNA, 1 flglfll sonicated
salmon DNA,
- 1 flglfll bovine serum albumins (BSA), and 10% dextran
sulfate.
- ISHR8: 100 fll dithiothreitol (DTT) solution (1.2 M)
- ISHR9: 800 ml NTE buffer containing 0.5M NaCl, 10 mM
Tris-HCI (pH 8.0), and 1 mM ethylene diaminse tetra acetate sodium salt (2NaEDTA)
- ISHRlO: Two 0.4 ml solutions containing RN ase A solution
(10 mg/ml RNase A).
- ISHR11: Two 600 ml solutions containing O.lxSSC, 15 mM
NaCI and 15mM sodium.
Equipment Disposable gloves, two forceps, two standard slide glass carriers
and supplies (20 slots), 29 rectangular staining dishes, 500 ml beaker, a starto be pre- ring bar, stirrer, clamp, pipettes, 200 ml and 1 I measuring cypared linder, Parafilm, incubator, rectangular culture dishes, 8 glass
rods, 1 ml micropipettes, and yellow tips
Reagents Fixative (50 m14% paraformaldehyde):
- 10 ml of 20% paraformaldehyde (use within a month). (To
prepare 20% paraformaldehyde, warm water and paraformaldehyde powder at 50-60°C and add ION NaOH until solution
becomes clear.)
- 40 ml of PB (final 0.07-0.08 M)
- PB: solution A: 0.2 M NaH2P0 4 • H 2 0 (27.6 gil)
- solution B: 0.2 M NaH2P04 • 2 H20 (35.6 gil)
- 23 ml solution A + 77 ml solution B + 100 ml H20 = 200 ml
(0.1 M)
- PBS: 5.84 g NaCl + 1.44 g NaH2P04 • 2 H20 + 0.20 g NaH2P04
in 1000 ml water, 450 ml formamide, 920 ml ethanol, 200 ml
xylene