Dopamine Receptor Binding and Quantitative Autoradiographic Study - Drugs of Abuse: Neurological Reviews and Protocols - page 293

2. Materials
2.1. Radioligand Binding Assays
2.1.1. Membrane Homogenate Preparation

1. Brain tissue: Fresh or fresh-frozen and stored at -70°C. Caudate-putamen is
most commonly used for D 1 -like and D 2 -like receptors, ventral striatum (nucleus
accumbens and olfactory tubercles) for D 3 .

2. Homogenizer (e.g., Brinkman Polytron, Tekmar Tissuemizer, or PRO).

3. Centrifuge tubes.

4. High-speed, refrigerated centrifuge.

5. Assay buffers. Store at 4°C for up to 2 wk.

a. [ 3 H]SCH 23390: 50 m M Tris-HCl, 120 m M NaCl, 5 m M KCl, 2 m M CaCl 2 ,
1 m M MgCl 2 , pH 7.4 at 23°C.

b. [ 3 H]Spiperone: 50 m M Tris-HCl, 5 m M KCl, 2 m M CaCl 2 , 1 m M MgCl 2 ,
pH 7.4, at 23°C.

c. [ 3 H]PD 128907: 50 m M Tris-HCl, 1 m M EDTA, pH 7.4, at 23° C.

2.1.2. Binding Assay

1. Radioligands:

a. D 1 -like receptors: [ 3 H]SCH 23390 (Amersham) ( see Note 2 ).

b. D 2 -like receptors: [ 3 H]Spiperone (Amersham) ( see Note 3 ).

c. D 3 receptors: [ 3 H]PD 128907 (Amersham) ( see Note 4 ).

2. Assay buffers. Store at 4°C for up to 2 wk.

a. [ 3 H]SCH 23390: 50 m M Tris-HCl, 120 m M NaCl, 5 m M KCl, 2 m M CaCl 2 ,
1 m M MgCl 2 , pH 7.4, at 23°C.

b. [ 3 H]Spiperone: 50 m M Tris-HCl, 5 m M KCl, 2 m M CaCl 2 , 1 m M MgCl 2 ,
pH 7.4, at 23°C.

c. [ 3 H]PD 128907: 50 m M Tris-HCl, 1 m M EDTA, pH 7.4, at 23°C.

3. Competing ligands (Sigma). A 1 m M stock may be prepared in ethanol and
stored at -20°C.

a. [ 3 H]SCH 23390: (+)-Butaclamol.

b. [ 3 H]Spiperone: (+)-Butaclamol.

c. [ 3 H]PD 128907: Spiperone.

4. Membrane homogenate.

75 Polystyrene culture tubes.
6. Cell harvester ( see Note 5 ).
7. Whatman GF/B fi lters.
8. Wash buffer: 50 m M Tris-HCl, pH 7.4, at 23°C. Store at 4°C.
9. Scintillation cocktail suitable for use with fi lters.
10. Scintillation vials.
11. Scintillation counter.
12. Protein assay kit.
13. Radioligand binding analysis software.

5. 12 ×