Alexa fluor 594 conjugated affinipure goat anti rabbit igg

Rats were perfused transcardially with 200 mL heparinized saline and 200 mL freshly prepared 4% paraformaldehyde through the ascending aorta under pentobarbital sodium anesthesia (50 mg/kg, i.p.). The brains were carefully removed, post-fixed for 12 h in 4% paraformaldehyde at room temperature, and dehydrated with 20% sucrose overnight at 4 °C. Afterward, they were placed in 30% sucrose for further dehydration overnight at 4 °C. Next, 30 μm-thick frozen coronal sections containing RVLM were cut using a cryostat (Thermo Scientific, USA). The sections were permeabilized with 0.3% Triton X-100 for 10–15 min, blocked with QuickBlock blocking buffer (Beyotime, China) for 30 min, and incubated with rabbit monoclonal c-Fos (9F6, 1:1000, Cell Signaling Technology, USA) and mouse monoclonal tyrosine hydroxylase (TH, F-11, 1:100, Santa Cruz) primary antibodies overnight at 4 °C. The next day, the sections were washed with phosphate buffered saline (PBS) and incubated with Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L; 1:400, Jackson ImmunoResearch, USA) and fluorescein isothiocyanate-conjugated AffiniPure Goat Anti-Mouse IgG (H + L; 1:200, Jackson ImmunoResearch, USA) secondary antibodies for 2 h at room temperature. A confocal laser scanning microscope (Zeiss, Germany) was used to monitor the fluorescent signals.