Ab109105

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.

Mice brain tissues containing cortex and hippocampus were collected before being fixed in 4% PFA for 12 h. The samples were dehydrated and then embedded in wax. A microtome (RM2235, Leica, Germany) was used to acquire 5 μm sections. For IHC staining, the sections were incubated with Anti-Synaptophysin antibody (ab32127, 1:200 dilution, Abcam, USA), Anti-SNAP25 antibody (ab109105, 1:200 dilution, Abcam, USA), Anti-PSD95 antibody (ab18258, 1:200 dilution, Abcam, USA) before antigen recovery. The three targeted proteins were visualized using 3, 3 – diaminobenzidine (DAB) kits (CW2069, CWBio, China). Images were obtained using a microscope (CX23, Olympus, Japan). Three mice per group and 3 serial sections per mouse were conducted in the experiment.
Nissl staining was performed according to the manufacturer’s protocols of the staining kit (G1434, Solarbio life science, Beijing, China). Briefly, the 5 μm brain sections were stained with methylene blue solution for 10 min before placed in Nissl differentiation solution for 3 sec. Sections were dehydrated in 100% alcohol. Images were acquired using a microscope (CX23, Olympus, Japan). Three mice per group and 3 serial sections per mouse were conducted in the experiment.