2.5.1. Single Immunohistochemical Labeling

Free-floating brain sections were pre-treated for 15 min with 3% H₂O₂, rinsed in 0.1 M PBS and then incubated in the primary antibody (Table 1) for 24–48 h at room temperature. After being rinsed in PBS, the sections were incubated for 1 h in the appropriate secondary biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) (Table S1) diluted 1:500 in PBS-TX. The sections were washed again with PBS and incubated for 1 h in peroxidase-conjugated streptavidin (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:2000 in PBS-TX. Peroxidase activity was developed with 0.05% 3,3´-diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO, USA) in the presence of 0.02% H₂O₂ and 0.08% nickel ammonium sulfate. Sections were mounted on gelatin-coated slides, air dried, dehydrated, and coverslipped with DPX mounting medium.