Mouse anti glua1

For western blotting, the procedure was performed as previous study at the age of 6 months [25 (link)]. Analysis of the data was performed using NIH ImageJ software, and the mean density of each band was normalized to β-actin signal in the same sample and averaged. For primary antibodies, we used mouse anti-β-actin (1:3000, Thermo, MA5–15739), rabbit anti-Tau (phosphor-S396) (1:1000, Abcam, ab109390), mouse anti-GluA1 (1:1000, Santa Cruz, sc-13,152), goat anti-GluA2 (1:200, Santa Cruz, sc-7611), mouse anti-GluN1 (1:1000, BD, 556308), rabbit anti-GluN2A (1:1000, Millipore, ab1555P) mouse anti-GluN2B (1:1000, BD, 610417).

Immunofluorescence staining was conducted as described previously (Fachim et al., 2016 (link); Zhang et al., 2017 (link)). The following primary antibodies were used: rabbit anti-OLFM3 (1:50, Proteintech, Wuhan, China), mouse anti-glial fibrillary acidic protein (GFAP) (1:50, Boster Bioengineering, Wuhan, China), guinea pig anti-microtubule-associated protein 2 (MAP2) (1:200, Sysy, Göttingen, Germany), guinea pig anti-vGlut1(1:200, Sysy, Göttingen, Germany), mouse anti-PSD95 (1:100, Abcam, United States), mouse anti-GluA1 (1:50, Santa Cruz Biotechnology, United States), mouse anti-GluA2 (1:100, Abcam, United States), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:50, Zhongshan Golden Bridge, Inc., Beijing, China), Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:200, Zhongshan Golden Bridge Inc., Beijing, China), and Alexa Fluor 633-conjugated goat anti-guinea pig IgG antibody (1:50, Abcam, United States). Finally, the samples were treated with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO, United States) for 10 min to identify the nuclei. Immunofluorescently labeled sections were imaged using a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and an Olympus IX 70 inverted microscope (Olympus America, Melville, NY, United States).

Western blotting was performed as described in our previous study^{36 (link)}. Briefly, hippocampus from GH mice and 4-week SI mice were dissected, homogenized, and solubilized at 4 °C for 1 h in lysis buffer (1% CHAPS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 50 mM NaF, 1 mM Na₃VO₄, and protease inhibitors, pH 7.2). Bound proteins were separated by SDS PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies: goat anti-EphB1 (1:500, Santa Cruz Biotechnology, sc-68317), goat anti-EphB2 (1:1000, R&D, P54763), mouse anti-synaptophysin (1:1000, Abcam, ab8049), rabbit anti-PSD95 (1:1000; Cell Signaling Technology, 3450), mouse anti-β-actin (1:3000; Thermo Fisher Scientific, MA5-15739), rabbit anti-GluN1 (1:1000, BD, 556308), mouse anti-GluA1 (1:1000, Santa Cruz Biotechnology, sc-13152), GluA2 (1:1000, Santa Cruz Biotechnology, sc-7611), GluA6 (1:1000, Santa Cruz Biotechnology, sc-7618), rabbit anti-GluN2A (1:1000, Millipore, ab1555P), and mouse anti-GluN2B (1:1000, BD, 610417).