Irdye 800cw or 680rd labelled secondary antibodies

Temporal cortices were lysed in RIPA buffer and spun, and the supernatants were quantified via BCA assay, and five μg of protein per temporal cortex lysate was resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF) for Western blotting. Membranes were blocked for 1h in Intercept (TBS) Protein-Free Blocking Buffer (LI-COR Bioscience, Lincoln, NE, USA) followed by incubation with anti-PSD95 (1:000, ab 238135; Abcam, Cambridge, MA, USA) and synaptophysin (1:000, ab52636; Abcam, Cambridge, MA, USA) antibodies overnight at 4 °C. Near-infrared-labeled secondary antibodies were used to detect antibody binding using IRDye^®, 680RD-, or 800CW-labelled secondary antibodies 1:15,000 (LI-COR Bioscience, Lincoln, NE, USA). Blots were scanned using a LI-COR Odyssey imaging system (LI-COR Bioscience, Lincoln, NE, USA). Band intensity values were normalized to their respective loading control, glyceraldehyde-3-phospate dehydrogenase (GAPDH) (sc32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Band intensities were quantified using ImageJ software. A band size was selected that fit the entirety of each band on the blot. The software was used to measure band intensity in each lane, which was normalized to its relevant loading control, GAPDH values, from the same membrane.