In Situ Hybridization with Isotopic Riboprobes for Detection of Striatal Neuropeptide mRNA Expression After Dopamine Stimulant Administration - Drugs of Abuse: Neurological Reviews and Protocols - page 117

2.2. Brain Section Pretreatment

1. Diethyl pyrocarbonate (DEPC; Sigma) distilled water: 0.1% in distilled water,
mix well and let stand overnight in a fume hood; autoclave for 2 h.

2. 10X Phosphate-buffered saline (PBS), pH 7.4 (Life Technologies, Invitrogen).

3. 4% Paraformaldehyde in 1X PBS, pH 7.4.

4. Proteinase K (Sigma).

5. Triethanolamine HCl (TEA; Sigma).

6. Acetic anhydride (Sigma).

7. Chloroform Normapur Analytical (Sigma).

8. Ethanol (Merk Eurolab).

2.3. Probe Preparation

1. [ 35 S-UTP ( 33 P; PerkinElmer Life Sciences or Amersham Biosciences).

2. 2.5 m M UTP (equal volumes, e.g., 5 µ L, of 10 m M AT P, CTP, and GTP (Life

Technologies).

3. RNA polymerase (e.g., SP6, T7, or T3; Life Technologies).

4. 1 U/ µ L of DNase I (Life Technologies).

5. 40 U/ µ L of RNase inhibitor (Life Technologies, Invitrogen).

6. Spin column (MicroSpin S-200 HR Columns; Amersham Biosciences).

7. DL -Dithiothreitol (DTT; Sigma).

2.4. Hybridization

1. 20X Standard sodium citrate buffer (SSC; Life Technologies; 1X = 3 M sodium
chloride, 0.3 M sodium citrate, pH 7.0).

2. Hybridization buffer: 1 mg/mL of sheared salmon testes DNA (Sigma),
500 g/mL of yeast tRNA (Sigma), 2X Denhardt's solution (Sigma), 20%

dextran sulfate (Amersham Biosciences), and 8X SSC.

µ

3. Formamide (Sigma).

4. DTT and DEPC distilled water.

2.5. Post-hybridization

1. RNase A (Ribonuclease A; Sigma).

2. RNase A buffer: 0.5 M NaCl, 0.04 M Tris-HCl, pH 8.0, and 1 m M EDTA.

3. Ammonium acetate (Sigma).

4. SSC, DTT, and ethanol.

2.6. Autoradiography

1. Kodak Biomax MR fi lm (Amersham Biosciences).

2.

14 C polymer standards (American Radiolabeled Chemicals Inc.).

3. D-19 Developer (Kodak).

4. Rapid Fix (Kodak).

5. Acetic acid (Sigma).