PV-immunoreactive, SST-immunoreactive, NPY-immunoreactive, and CB-immunoreactive interneurons in the hippocampus were quantified in sections processed for immunoperoxidase staining. Free-floating sections were washed three times for 10 min each with Tris-Triton buffer, pH 7.4 followed by incubation with rabbit primary antibody against PV (Table 1) in a solution containing 2% Triton X-100 and 2% normal goat serum (NGS) in Tris-Triton buffer, pH 7.4, overnight at 4°C under continuous agitation. The next day, the sections were rinsed again three times for 10 min each with Tris-Triton buffer, pH 7.4 before being incubated at room temperature (RT) for 30 min with the biotinylated secondary antibody (goat anti-rabbit, Jackson ImmunoResearch, 1:300) in a solution containing 2% NGS in Tris-Triton buffer, pH 7.4. After another washing step of three times 10 min, the sections were incubated for 30 min in avidin-biotin complex solution (Vectastain Elite kit; Vector Laboratories) and rinsed again three times for 10 min. To allow equal penetration of the tissue, the sections were preincubated in diaminobenzidine (DAB) solution (0.5 × g/L DAB in Tris-Triton buffer, pH 7.7) for 5 min under agitation, before the reaction was started by adding 2 ml of DAB solution containing 0.01% hydrogen peroxidase. After 5–7 min, depending on the intensity of the staining, the reaction was stopped by transferring the sections into ice-cold PBS followed immediately by another washing step of three times 10 min in PBS. The sections were mounted on gelatin-coated glass slides and left to dry overnight. On the following day, they were dehydrated in ethanol of increasing concentrations (2 × 70%, 2 × 96%, 3 × 100%) for 5 min each followed by clearing in xylene four times for 5 min. Finally, the sections were cover-slipped with Eukitt mounting medium (Merck).

Primary antibodies used for immunohistochemistry

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