Rabbit anti c fos and mouse anti ngf antibodies

To detect c-Fos expressions in the vlPAG, PMC, and spinal cord (L5) regions, c-Fos and NGF immunohistochemistry was performed as previously described [20 (link)]. Briefly, tissue sections were incubated overnight with rabbit anti-c-Fos and mouse anti-NGF-antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at a dilution of 1:1,000. The sections were then incubated for 1 hour with anti-rabbit and anti-mouse secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA). The sections were subsequently incubated with an avidin–biotin–peroxidase complex (1:100; Vector Laboratories) for 1 hour at room temperature. Immunoreactivity was visualized by incubating the sections in a solution consisting of 0.02% 3,3´-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 0.03% H₂O₂ in 50mM Tris–HCl (pH, 7.6). The sections were then washed 3 times with PBS and mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount (Fisher Scientific, Waltham, MA, USA).

An immunohistochemical analysis was conducted to evaluate c-Fos and NGF expression in the MPA, vlPAG, PMC, and spinal cord L4–5 regions, following a previously described method [10 (link),12 (link)]. Free-floating tissue sections were incubated overnight with rabbit anti-c-Fos and mouse anti-NGF antibodies (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the sections were incubated for 1 hour with biotinylated anti-rabbit c-Fos and anti-mouse NGF secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA). Next, the sections were incubated with avidin-biotin-peroxidase complex (Vector Laboratories) for 1 hour at room temperature. For staining, the sections were incubated in 0.03% DAB and 0.03% hydrogen peroxide for 5 minutes. The sections were mounted onto gelatincoated slides. The slides were air-dried overnight at room temperature, and coverslips were mounted using Permount (Fisher Scientific, Waltham, MA, USA).