Biotinylated goat anti rabbit igg

Frozen coronal sections were cut 30 mm thick. After being rinsed in 0.01 M of PBS, they were incubated with blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) for 30 min and then with rabbit anti-Fos antibody (sc-52, Santa Cruz, CA, USA) at a dilution of 1:2000 for 24 h at 44 °C. Subsequently, the sections were incubated with the biotinylated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA, USA) for 1 h at room temperature. The specificities of the staining were tested on the sections in the control rats by omitting the primary antibodies.

Rats were perfused with 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 250 mL freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (4°C). The brains were removed immediately, postfixed for 4 hours, and dehydrated overnight in 20% sucrose in 0.1 M phosphate buffer saline (PBS) at 4°C. Thirty-micrometer successive coronal sections containing the VLPAG were cut and then collected into 0.01 M PBS. Endogenous active enzymes were blocked with 2% goat serum in 0.01 M PBS containing 0.3% Triton X-100 for 1 hour at room temperature. The GFAP-immunoreactive (GFAP-IR), c-Fos-immunoreactive (c-Fos-IR), and p-ERK1/2-immunoreactive (p-ERK 1/2-IR) sections were incubated overnight at 4°C with rabbit anti-GFAP 1:300 (Bioss Antibodies, Beijing, China), rabbit anti-c-Fos (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-p-ERK1/2 (1:1000; Cell Signaling Technology, Danvers, MA, USA). Subsequently, the sections were incubated with biotinylated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA, USA) for 1 hour at room temperature. The specificities of the staining were tested on the sections in the control rats by omitting the primary antibodies.