8 In Situ Hybridization for RNA: Radioactive RNA Probe
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Materials
- Tissue sections for a positive control: 4 glass slides with sec- Contents in
tions of submandibular glands from adult male mouse (C3H). the ISH kit
- Solutions for preparation of RNA probe
- RNA synthesis kit
- RNA polymerase buffer 20 ~l
- 10 mM rATP 5~1
- 10 mM rCTP 5 ~l
- 10 mM rGTP 5 ~l
- 10 mM rUTP 5 ~l
- RNase inhibitor 5 ~l
- SP6 RNA polymerase 5 ~l
- T7 RNA polymerase 5 ~l
- T3 RNA polymerase 5 ~l
- DNase I (5 mg/ml) 5 ~l
- Sterilized water 500 ~l
- Composition of RNA polymerase buffer
- 200 mM Tris-HCl (pH 7.5)
- 30 mM MgClz
- 25 mM NaCl
- 10 mM Spermidine-(HClh
- 50 mM DTT
- 0.5 mg/ml BSA
- Solutions for ethanol precipitation
- Ethanchinmate 10 ~l
- 3M Sodium acetate (pH 5.2) 100 ~l
- Alkaline hydrolysis buffer 800 ~l containing 40 mM NaHC03
and 60 mM NazC03 (pH 10.2).
- Linearlized DNA template for RNA probe synthesis
- A plasmid containing a fragment of mouse nerve growth
factor (NGF) cDNA
- For antisense probe 2 ~l (1 ~1!ml)
- For sense probe (as a negative control) 2 ~l (1 ~1!ml)
- Solutions for ISH for two sets (20 slide glasses/set)
- ISHRl: two 600 ml PBS buffers containing 0.1 M NaCl and
10 mM sodium phosphate (pH 7.4)
- ISHR2: 800 ml PBS/glycine buffer containing 2mg/ml glycine, 0.1 M NaCl and 10 mM sodium phosphate (pH 7.4)
- ISHR3: 800 ml acetylation buffer containing 0.1 mM
triethanolamine (pH 8.0)