13 In Situ Hybridization for RNA: Nonradioactive Probe: Oligo-DNA Probe
185
RNase-free glassware, autoclaved water and chemicals for molecular biology use, if possible. All solutions, except those containing Tris, should be treated with 0.1 % DEPC followed by autoclaving.
- 3-aminopropyltriethoxysilane (APS)-coated slide glasses:
Soak slide glasses in acetone dissolved with APS for 5 to 10
seconds. Wash them briefly with acetone and DEPC-treated
water sequentially. Dry the slides overnight. Commercial
APS-coated slide glasses are also available (Matsunami,
Tokyo or DAKO)
- 4% paraformaldehyde in PBS:
Add 40g paraformaldehyde to warm 800 ml of DE PC-treated
water on a heat plate. Heat the solution and add a few drops of
1M NaOH. When paraformaldehyde dissolves completely, the
color of the solution changes from cloudy to clear. Add 100 ml
of 0.1 M PBS to the solution and leave to cool in ice. Then,
check pH and adjust to pH 7.4 by 1M HCl.
Note: Paraformaldehyde can be dissolved only in warm alkaline
solution.
Note: Preparation should be completed under the laboratory
hood.
- 50 x Denhardt's solution:
1 % bovine serum albumin (Fraction V; Sigma), 1% Ficoll
(Type 400, Pharmacia) and 1 % polyvinylpyrrolidone in
DEPC-treated water.
- Standard saline citrate (SSC):
20x SSC; 3 M NaCl + 0.3 M trisodium citrate
- Formamide:
Formamide for molecular biology is available from several
chemical firms. However, if any yellow color is present, the
formamide should be deionized by resin. After filtering,
deionized formamide can be stored at -70°C.
- Prehybridization buffer:
- 4x SSC
- 50 mM sodium phosphate
- 5x Denhardt's solution
- 0.2 mg/ml salmon testis DNA
- 0.2 mg/ml yeast tRNA
- 20% deionized formamide
185
RNase-free glassware, autoclaved water and chemicals for molecular biology use, if possible. All solutions, except those containing Tris, should be treated with 0.1 % DEPC followed by autoclaving.
- 3-aminopropyltriethoxysilane (APS)-coated slide glasses:
Soak slide glasses in acetone dissolved with APS for 5 to 10
seconds. Wash them briefly with acetone and DEPC-treated
water sequentially. Dry the slides overnight. Commercial
APS-coated slide glasses are also available (Matsunami,
Tokyo or DAKO)
- 4% paraformaldehyde in PBS:
Add 40g paraformaldehyde to warm 800 ml of DE PC-treated
water on a heat plate. Heat the solution and add a few drops of
1M NaOH. When paraformaldehyde dissolves completely, the
color of the solution changes from cloudy to clear. Add 100 ml
of 0.1 M PBS to the solution and leave to cool in ice. Then,
check pH and adjust to pH 7.4 by 1M HCl.
Note: Paraformaldehyde can be dissolved only in warm alkaline
solution.
Note: Preparation should be completed under the laboratory
hood.
- 50 x Denhardt's solution:
1 % bovine serum albumin (Fraction V; Sigma), 1% Ficoll
(Type 400, Pharmacia) and 1 % polyvinylpyrrolidone in
DEPC-treated water.
- Standard saline citrate (SSC):
20x SSC; 3 M NaCl + 0.3 M trisodium citrate
- Formamide:
Formamide for molecular biology is available from several
chemical firms. However, if any yellow color is present, the
formamide should be deionized by resin. After filtering,
deionized formamide can be stored at -70°C.
- Prehybridization buffer:
- 4x SSC
- 50 mM sodium phosphate
- 5x Denhardt's solution
- 0.2 mg/ml salmon testis DNA
- 0.2 mg/ml yeast tRNA
- 20% deionized formamide