Alexa 568 conjugated anti rabbit igg

After physiological experiments, mice were deeply anesthetized with isoflurane or urethane and transcardially perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). The brain was removed and post-fixed in 4% PFA at 4 °C for one day. Coronal sections of 50–100 µm thickness were obtained using a microslicer (DTK-1000N, Dosaka EM). For immunofluorescence, the sections were incubated at room temperature in PBS (5 min × 3) and in PBS containing 0.3% Triton X-100 (PBST) for 5 min. The sections were further incubated in the blocking solution (5% normal goat serum, 1% bovine serum albumin, and 0.3% Triton X-100 in PBS) for 60 min at room temperature. We then incubated the sections with a rabbit polyclonal antibody to parvalbumin (1:2000, Abcam) in the blocking solution at 4 °C overnight, washed with PBST (5 min × 3), and incubated with Alexa 568-conjugated anti-rabbit IgG (1:500, Abcam) for 2 h at room temperature. The sections were mounted on glass slides, and the fluorescence images were taken with an inverted fluorescence microscope (BZ-X800, Keyence).