Mouse brains were perfusion-xed in 4% paraformaldehyde and embedded para n and cut into 4 micrometer-thick sections used for the immunohistochemical analysis of monoclonal antibodies for DCX (E-6 monoclonal, 1:50, Santa Cruz Biotechnology Inc., Dallas, USA), MAP2ab (AP-20 monoclonal, 1:100, SIGMA-ALDRICH, St. Louis, USA), GluA1 (AB1540 polyclonal, 1:100, EMD Millipore Corp., Burlington, USA), and GluA2 (AB1768-1 polyclonal, 1:5, EMD Millipore Corp., Burlington, USA), respectively.
Quantitative immunostaining area by ZEISS ZEN Intellesis software.
Acquisition of immunostained area in each MAP2, GluA1, and GluA2 expression area was performed using AxioVision (Carl Zeiss, Germany) microscopy (20X objective lens) with a ZVI format. Generating data image processing with image segmentation was performed using machine learning with ZEN Intellesis software. More than 1000 cells were examined in each group. One-way ANOVA was used to test the variance among the three groups, excluding data of 3SD or more. Then, we used the following sample numbers: HFD series: MAP2 CD n=731, HFDs n=288, HFD+PER n=225;GluA1 CD n=220, HFD n=156, HFD+PER n=94; GluA2 CD n=78, HFD n=114, HFD+PER n=182; GluA2/GluA1 CD n=78, HFD n=94, HFD+PER n=94. The Bonferroni method was used for post-hoc testing to analyze signi cant differences among the groups.
Quantitative immunostaining area by ZEISS ZEN Intellesis software.
Acquisition of immunostained area in each MAP2, GluA1, and GluA2 expression area was performed using AxioVision (Carl Zeiss, Germany) microscopy (20X objective lens) with a ZVI format. Generating data image processing with image segmentation was performed using machine learning with ZEN Intellesis software. More than 1000 cells were examined in each group. One-way ANOVA was used to test the variance among the three groups, excluding data of 3SD or more. Then, we used the following sample numbers: HFD series: MAP2 CD n=731, HFDs n=288, HFD+PER n=225;GluA1 CD n=220, HFD n=156, HFD+PER n=94; GluA2 CD n=78, HFD n=114, HFD+PER n=182; GluA2/GluA1 CD n=78, HFD n=94, HFD+PER n=94. The Bonferroni method was used for post-hoc testing to analyze signi cant differences among the groups.