Plan apo vc 20x

One series of sections were used for this immunostaining. After several PBS washes, sections were incubated in the following reagents: biotinylated Wisteria floribunda agglutinin (WFA), rabbit anti-parvalbumin antibody and guinea-pig anti-c-Fos antibody diluted in PBS containing 0.25% Triton-X100 and 1% bovine serum albumin (BSA) for 24 h at 20 °C (see Table 1). After being washed with PBS, the sections were incubated in a mixture of DyL405-conjugated donkey anti-goat IgG, Alexa 488-conjugated streptavidin, Cy3-conjugated goat anti-guinea pig IgG and Alexa647-conjugated donkey anti-rabbit IgG with 0.2% Triton-X100 for 2 h at 20 °C (see Table 2). After being washed with PBS, the sections were mounted on glass slides and coverslipped using Mowiol 4–88 fluorescent mounting medium and analyzed using Nikon C2 confocal microscope (Plan Apo VC 20x, NA = 0.75; xy, 1.24 μm/pixel; z-step size, 2 μm). Overview images (tiles) of orbitofrontal and prefrontal cortices were acquired by 405, 488, 561, and 642 nm lasers. Images were taken at levels 2.68−1.42 mm anterior from bregma. For the PV+/PNN+/c-Fos co-expression quantification, single-labeled, double-labeled and triple-labeled neurons were counted manually using NIS Elements Software (Nikon Europe; RRID:SCR_014329).