c-Fos immunohistochemistry

The brains were dissected out 1 h after ICV injection with 3 × 10⁻⁶ M AngII alone (n = 5), or 3 × 10⁻⁵ M ANP in the presence of AngII (n = 5). The procedure for the immunohistochemistry was previously described^21,52. The brains were fixed in 4% paraformaldehyde in phosphate buffer (PB) and embedded in Paraplast (McCormick Scientific, Richmond, IL, USA). Serial sections were prepared at 7 μm and mounted onto MAS-coated slides (Matsunami Glass, Osaka, Japan). The sections were deparaffinized, rehydrated and then rinsed in phosphate buffered saline (PBS). The sections were immersed in methanol containing 0.3% H₂O₂ for 30 min at room temperature. The sections were rinsed thoroughly in PBS after the antigen activation and then pretreated with a blocking solution (2% normal goat serum, 0.01% NaN₃ in PBS) for 1 h at room temperature and incubated with a polyclonal antibody raised against human c-Fos (1:200, sc-253, SantaCruz, Dallas, TX, USA) for 12 h at 4 °C. Specificity of the antibody against c-Fos protein of the mudskipper was previously confirmed by Western blotting analysis⁵². After rinsing in PBS, the sections were treated with ABC Elite kit (PK-6101, Vector, Burlingame, CA, USA) according to the manufacturer’s instruction. The sections were rinsed in 0.1 M PB and immersed in 0.01% 3,3′-diaminobenzidine tetrahydrochloride (DAB) in PB for 3 min to intensify colorization and then incubated in 0.01% DAB solution containing 0.01% H₂O₂ for 3 min in dark. Finally, the sections were rinsed in PB and distilled water, and immunoreactive c-Fos signals were examined and photographed using a digital camera (DMX1200; Nikon, Tokyo, Japan). The photomicrographs were binarized by graphic software Image J (https://imagej.nih.gov/ij/) and the number of c-Fos positive neurons was counted manually and compared between the cohorts. Every third section was analysed in order not to count positive cells repeatedly.