The PND30–35 rats were deeply anesthetized with sevoflurane and transcardially perfused
with phosphate buffered saline (PBS) (pH 7.4) followed by 4% formaldehyde (pH 7.4). The
hippocampus was collected and postfixed with the same fixative for 24 h at 4 °C. The
hippocampus was cut into a thickness of 30 μm coronal sections on a freezing microtome.
The sections were blocked in 3% bovine serum albumin (BSA) and 0.3% Triton X-100
(Sigma-Aldrich) for 1 h at room temperature followed by incubation with primary antibodies
as required (mouse anti-SK2 (1:1000 Millipore,); rat anti-NeuN (1: 1000, Abcam)) in 1% BSA
overnight at 4 °C. After the sections were washed, they were incubated for 1 h with
secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:500; Alexa Fluor 594 goat
anti-rabbit IgG, 1:500; both from Cell Signaling Technology) and 4′,
6-diamidino-2-phenylindole (DAPI) solution for 10 min at 37 °C. Fluorescence was detected
using a confocal laser microscope (LSM 800, Zeiss). The optical density of neuropeptide Y
(NPY) was measured using ImageJ software (National Institutes of Health).
with phosphate buffered saline (PBS) (pH 7.4) followed by 4% formaldehyde (pH 7.4). The
hippocampus was collected and postfixed with the same fixative for 24 h at 4 °C. The
hippocampus was cut into a thickness of 30 μm coronal sections on a freezing microtome.
The sections were blocked in 3% bovine serum albumin (BSA) and 0.3% Triton X-100
(Sigma-Aldrich) for 1 h at room temperature followed by incubation with primary antibodies
as required (mouse anti-SK2 (1:1000 Millipore,); rat anti-NeuN (1: 1000, Abcam)) in 1% BSA
overnight at 4 °C. After the sections were washed, they were incubated for 1 h with
secondary antibodies (Alexa Fluor 488 goat anti-mouse IgG, 1:500; Alexa Fluor 594 goat
anti-rabbit IgG, 1:500; both from Cell Signaling Technology) and 4′,
6-diamidino-2-phenylindole (DAPI) solution for 10 min at 37 °C. Fluorescence was detected
using a confocal laser microscope (LSM 800, Zeiss). The optical density of neuropeptide Y
(NPY) was measured using ImageJ software (National Institutes of Health).