Viral-Mediated Gene Transfer to Study the Behavioral Correlates of CREB Function in the Nucleus Accumbens of Rats - Drugs of Abuse: Neurological Reviews and Protocols - page 325

2.2. Titering the Vector

1. Laminar fl ow hood and 37°C humidifi ed incubator with 10% CO 2 atmosphere.

2. Tissue culture microscope and hemocytometer.

3. 24-Well tissue culture dishes.

4. PC12 cell line derived from rat pheochromocytoma (adrenal cells with nerve
growth factor [NGF] reponsiveness).

5. DMEM + 10% horse serum (HS) + 5% FBS.

6. Phosphate-buffered saline (PBS): prepare a 10X PBS stock by mixing l g of
KH 2 PO 4 , 10.8 g of Na 2 HPO 4 •7H 2 O, 1 g of KCl, 40 g of NaCl, and water to 500 mL
fi nal volume. The pH should be approx 7.0. Filter-sterilize, and store at room
temperature (RT). Dilute 10-fold before use.

7. Poly- D -lysine (PDL): Prepare 1 mg/mL of PDL (PDL, mol wt 70,000-150,000
Sigma P6407) in water, fi lter sterilize, and store at -20°C. This is a 50X stock.
Dilute to 20 g/mL in water immediately prior to use.

8. Tris-buffered saline (TBS): 100 m M Tris-HCl, pH 7.5, and 150 m M NaCl. Store
at RT.
9. TBS-serum-Triton (TST): 1% goat serum and 0.1% Triton X-100 in TBS. Store
at 4°C.
10. Fe solution: Mix 200 mL of PBS, 332 mg of potassium ferricyanide, 424 mg of
potassium ferrocyanide, 0.2 mL of 1 M MgCl 2 , 0.2 mL of 20% Nonidet P-40,
and 0.2 mL of 10% sodium deoxycholate. Store at 4°C in a darkened container,
as this reagent is light sensitive.
11. X-gal (5-bromo-4-chloro-3-indolyl-

µ

- D -galactoside; a 50 mg/mL stock in
dimethyl sulfoxide can be stored in aliquots at -20°C).
12. 4% Paraformaldehyde: Add 20 g of paraformaldehyde to 300 mL of H 2 O and
heat to 55-60°C. Slowly add 1 M NaOH dropwise over about 10 min until the
solution becomes clear. Cool the solution to room temperature. Use pH paper
to check that the pH is 7.0-7.5 (add more NaOH if necessary). Add 100 mL of
0.5 M sodium phosphate buffer, pH 7.0, and then water to a fi nal volume of
500 mL. The fi nal pH should be 7.0-7.5. Store at 4°C.
13. Alkaline phosphatase (AP) buffer: 100 m M Tris-HCl, pH 8.5, 150 m M NaCl,
and 5 m M MgCl 2 .
14. AP substrate: Prepare 5-bromo-4-chloro-3-indolyl phosphate (BCIP) stock
solution by dissolving one tablet (Sigma B-0274) in 0.5 mL of 100% dimethyl
formamide. Prepare nitroblue tetrazolium (NBT) stock solution by dissolving
one tablet (Sigma N-5514) in 1 mL of water. Make substrate solution by adding
330 µ L of NBT stock to 10 mL of AP buffer. Mix, and then add 33 µ L of BCIP

stock. Store at -70°C in aliquots.

2.3. Cell Culture

1. Primary neuronal cultures.

2. Western immunoblotting: Rabbit anti-CREB antibody, 1 4000 (Cell Signaling),

goat peroxidase labeled antibody to rabbit IgG, 1 5000 (Vector Laboratories),

and chemiluminescence (Perkin Elmer).