Anti c fos rabbit polyclonal antibodies

Brain or spinal cord sections were removed after perfusion with 4% paraformaldehyde, postfixed and cryosectioned into 30 µm-thick sections at the level of the CN (coordinates: anterior, −14.30~−14.60 mm; lateral, ±1.0~1.4 mm; deep, −7.8~−8.2 mm), the NAc (coordinates: anterior, 1.60~1.30 mm; lateral, ±0.6~1.0 mm; deep, −6.8~−7.4 mm) or the LHb (coordinates: anterior, −3.5 mm; lateral, ±0.7 mm; deep, −4.9 mm). The sections were incubated overnight (approximately 16 hr) at 4 °C with anti-c-fos rabbit polyclonal antibodies (1:200; Sigma, USA) followed by a 2-hr incubation at room temperature with a biotinylated donkey anti-rabbit Alexa Fluor 594 (red; 1:500; Sigma, USA) and mounted onto gelatin-coated slides. The sections were photographed and quantified using confocal laser scanning microscopy (LSM700, Carl Zeiss, Germany).

In a separate set of animals, rats (n = 13) were randomly assigned into Normal (n = 5), Cocaine (cocaine injection only, n = 4) and Cocaine + PEPA (PEPA injection into LH in cocaine treated rats, n = 4) groups. Sixty minutes after cocaine and/or PEPA injection, brains were taken out after perfusion with 4% paraformaldehyde, postfixed, and cryosectioned at 30 μm thickness. The sections were incubated with anti-c-Fos rabbit polyclonal antibodies (1:500; Sigma, USA), followed by incubation of a biotinylated donkey anti-rabbit Alexa Fluor 594 (1:500; Sigma). The slices were mounted on gelatin-coated slides, photographed, and quantified using a confocal laser scanning microscope (LSM700, Carl Zeiss, Jena, Germany). The number of c-Fos labeled cells was counted in a blindly chosen 290 μm × 300 μm area of the LH. The 4–6 brain pieces per animal were analyzed and averaged. Data were expressed as number of positive cells per group.