Alexafluor 488 labeled anti rabbit igg antibody

Macrophages were cultured on chamber slides. They were untreated or treated with cocaine (10 μM), infected or uninfected with HIV-1 (10 ng/ml) for 72 h. They were fixed in 4% paraformaldehyde and blocked with 5% normal goat serum in PBS/Triton X100 (1 h). Cells were then incubated with primary antibodies overnight at 4 °C, washed thrice with PBS, and stained with AlexaFluor 546–labeled anti–mouse IgG antibody or AlexaFluor 488–labeled anti–rabbit IgG antibody (Molecular Probes®; Invitrogen) for 2 hours. Subsequently, cells were washed thrice with PBS, and slides were mounted using Prolong Gold antifade with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen). Slides were examined under a Zeiss 880 Meta confocal microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY), and images were acquired using ZEN2 software (Carl Zeiss).

Rats (n = 3/group) received an i.c.v. injection of ghrelin or ghrelin + LEAP2. After 90 min, they were anesthetized with a mixture of three anesthetics (Domitor, 1 mg/mL; midazole, 5 mg/mL; vetorphale, 5 mg/mL) in a volume of 0.1 mL/20 g body weight and transcardially perfused with ice-cold PBS and ice-cold 4% PFA. Hypothalamic sections (thickness, 40 µm) were cut on a freezing microtome (Yamato Kohki Industrial, Saitama, Japan). For Fos immunohistochemistry, free-floating sections were blocked in blocking buffer (3% normal serum in 0.3% PBS-T) for 60 min, and then incubated overnight at 4°C with rabbit anti-c-Fos (Santa Cruz Biotechnology). After washing with PBS, the sections were incubated for 60 min at room temperature with Alexa Fluor 488-labeled anti-rabbit IgG antibody (1:500; Invitrogen). Images were visualized by confocal microscopy. Fos-positive cells were counted manually.