Biotinylated goat anti rabbit

A subgroup of GAD65−/− mice and their GAD65+/+ littermates received transcardial perfusion with phosphate buffered 4% paraformaldehyde solution at pH 6.8, as previously described (Raza et al., 2017 (link)). After postfixation overnight in the same fixative and overnight immersion in wt/vol 20% sucrose at 4 °C, brains were cut into 30 μm thick, serial coronar sections at the level of the PFC, posterior ACC/dorsal striatum and dorsal hippocampus/amygdala, stored as free-floating sections in phosphate buffered saline at 4 °C and then incubated with a rabbit antibody against cFos (1:1000, Cell signaling #2250, Danvers, MA, USA) for 48 h. As secondary antibody biotinylated goat anti-rabbit (1:200, Jackson ImmunoResearch, Ely, UK) in phosphate buffer (PB) with 0.2% Triton (PBT) was used and labelled with Cy2 via Streptavidin (1:1000, Jackson ImmunoResearch, Ely, UK) in PB. Nuclei were visualized with DAPI (300 nM, Thermofischer, Waltham, MA, USA). Immunostainings were imaged using a DMI6000 epifluorescence microscope (Leica, Wetzlar, Germany) in both hemispheres from 2 slices per animal and region. cFos-positive cells were counted manually within the selected brain areas and cell density was normalized to area size measured with imageJ software. The cell counts per target area were averaged for each individual animal and used for statistical comparison between groups.