Ab238135

Hippocampal tissues were homogenized in RIPA cell lysate (Beyotime, P0013B), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. Each protein sample was loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel spiked wells. Electrophoresis was performed at constant pressure of 80 V for approximately 1 h. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), fixed with western closure solution (5% skim milk powder), slowly shaken on a shaker for 2 h at room temperature, and then incubated with the following primary antibodies: rabbit anti-postsynaptic density-95 (PSD-95) antibody (1:2000, abcam, ab238135), rabbit anti-synaptophysin (SYN) antibody (1:1000, Bioss, bs-8845R), rabbit anti-BDNF antibody (1:1000, abcam, ab108319), and rabbit tyrosine kinase receptor B (TrkB) antibody (1:5000, abcam, ab187041). The samples were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20000, Zsbio, ZB-2301) for 1.2 h. The membranes were washed with phosphate-buffered saline with Tween and the ECL luminescence kit (Thermo, 340,958) was used to detect the proteins. Finally, the intensity of the bands was analyzed by Image J software (Media Cybernetics, United States).

Rats (n = 4 per groups) were deeply anesthetized with 10% pentobarbital (10 mL/kg) and slowly perfused with 300 mL of phosphate buffer saline (PBS, pH 7.2), followed by 300 mL of 4% paraformaldehyde (PFA). The brain was removed and immobilized in 4% PFA for 72 h and then dehydrated in 30% sucrose for 72 h. Tissues on slides were treated with 0.2% Triton X-100 and blocked with 5% goat serum. The slide was incubated overnight at 4 °C with the following primary antibodies: Ki-67 (D3B5) Rabbit mAb (Ki-67, 9129, Cell Signaling); Anti-Doublecortin (DCX, ab254133, Abcam); Anti-Iba1 antibody (Iba-1, ab178846, Abcam); Anti-PSD95 antibody (ab238135, Abcam); Anti-CD68 antibody (ab125212, Abcam), Neuronal nuclei (E4M5P) Mouse mAb (NeuN, 94403, Cell Signaling). Anti-ZO1 tight junction protein antibody (ZO-1, ab221547, Abcam) and Anti-Claudin 1 antibody (Claudin-1, ab211737, Abcam). After the primary antibodies were rinsed with PBS, the tissues were covered with Alexa Fluor® 488- and Alexa Fluor® 594-conjugated fluorescent secondary antibodies and incubated in the dark for 60 min. After rinsing the secondary antibodies with PBS, 4,6-Diamidino-2-phenylindole (DAPI)solution was added, and the slides were incubated in the dark for 3–5 min. The slides were observed with an Olympus BX53 fluorescent microscope equipped with a DP74 Microscope Digital Camera.

Hippocampal tissues or cells were homogenized in buffer (0.5 mol/L Tris; 1% NP40; 1% Triton X-100; 1 g/L sodium dodecyl sulfate; 1.5 mol/L NaCl; 0.2 mol/L EDTA; 0.01 mol/L EGTA; and protease inhibitor and/or phosphatase inhibitor), sonicated, and incubated at −20°C for 20 minutes, followed by centrifugation at 12,000 g for 20 minutes at 4°C. The supernatant was collected and protein concentration determined by the bicinchoninic acid (BCA) method. Next, 50 μg protein were loaded on 10% SDS polyacrylamide gel. The primary antibodies used were anti-TRPC6 (Alomone, ACC-017), anti-PSD 95 (Abcam, ab238135), anti-SNAP25(Abcam, ab109105), anti-SYP (Abcam, ab32127), anti-MFN1 (Abcam, ab126575), anti-MFN2 (Abcam, ab124773), anti-Drp1(Abcam, ab184247), anti-p-Drp1(mice Ser 622; CST, 3455), anti-p-Drp1 (mice Ser 643; Abcam, ab193216), anti-GLUT1-5 antibody (Affinity, AF6731, DF7510, AF5463, AF5386, DF13545), anti-SGLT1 (Invitrogen, PA5-77460), and anti-SGLT2 (Abcam, ab137207), followed by incubation with the secondary antibodies (ZSGB-BIO). Protein expression was normalized to GAPDH intensity or total protein content. See complete unedited blots in the supplemental material.

Proteins from the basal forebrain or hippocampus were extracted according to the manufacturer's instructions (C500007, Sangon Biotech, China), and then separated by SDS‐polyacrylamide gel electrophoresis. After separation, the proteins were transferred onto polyvinyl difluoride membranes (10600023, GE, USA). After blocking, the membranes were incubated overnight at 4°C with rabbit anti‐BDNF (1:2000; ab108319, Abcam, USA), rabbit anti‐PSD95 (1:2000; ab238135, Abcam, USA), or GAPDH (1:20,000; ab181603, Abcam) antibody. On the following day, membranes were washed then incubated for 1 h at room temperature with Tris‐buffered saline containing 0.1% Tween 20 (TBST) and the horseradish peroxidase (HRP)‐conjugated secondary antibody IgG‐HRP (ab6721, Abcam). Densitometric analysis was performed using the BioRad Western blot detection system. GAPDH was used as the internal control.

The hippocampal tissues were harvested and fixed with 4% paraformaldehyde in PBS to investigate BDNF and PSD95 protein expression after finishing MWM. Then, hippocampal tissues sections (5 μm) from the brain were cut and blocked with Tris‐buffered saline containing 0.1% Tween 20 (TBST) and 5% goat serum for 1 h at room temperature. The slides were incubated overnight at 4°C with a rabbit anti‐BDNF antibody (1:200; ab108319, Abcam, USA) or PSD95 (1:100; ab238135, Abcam, USA). On the following day, sections were washed three times and incubated at room temperature with goat anti‐rabbit IgG H&L (1:5000, ab6717, Abcam, USA). Hippocampal slices from each mouse were analyzed using imaging observation equipment (LEICA DMI8, LEICA, Germany) to observe immunohistochemistry staining. Cases were scored as negative if no or only weak BDNF or PSD95 staining was observed. Three sections were obtained from each mouse (n = 4 in each group), and three visual fields were randomly selected and observed in each tissue section. The cells were counted, and the mean rates of positive cells from each mouse were compared between the aCSF group and ASO7 group. All quantitative analyses were performed by an experimenter blinded to the group assignment.

The brain slices were washed 3 times with PBS for 5 min each time and then treated with 0.1% Triton X-100 (Beyotime Biotechnology, ST795, Shanghai, China) diluted with PBS for 20 min. Then, slices were placed in 5% BSA (Beyotime Biotechnology, ST023, Shanghai, China) diluted with PBS and sealed at room temperature for 1 h. After incubating overnight with the primary antibody [rabbit antibody PSD95 (Abcam, ab238135, Cambridge, UK, 1:200), rabbit antibody IBA1 (Fujifilm, 019-19741, Tokyo, Japan, 1:300)] at 4 °C, the sections were washed 3 times with PBS. A secondary antibody [goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (Abcam, ab150077, 1:500)] was then applied at room temperature for 1 h, and the slices were washed in PBS 3 times. Finally, the nuclei were restained with 1 μg/mL DAPI (Beyotime Biotechnology, C1002, Shanghai, China) for 10 min, and the slides were mounted with an anti-fluorescence quenching seal. The slides were viewed with a fluorescence microscope (BioTek, Cytation5, Hong Kong, China) and all images were collected using a microscopic imaging system (BioTek, Cytation5, Hong Kong, China). The ImageJ (v1.54f) application was used to analyze the collected images.