Mouse anti synaptophysin

The primary antibodies used were: mouse anti-β-actin (1:1000, sc-47778, Santa Cruz Biotechnology, Inc.), goat anti-4HNE (1:1000, H6275-02, US Biological Life Sciences), mouse anti-Total OXPHOS Human WB Antibody Cocktail (1:1000, ab110411, Abcam, Inc.), rabbit anti-GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Inc.), mouse anti-human tau (1:1000, 2024-10-31, Dako), rabbit Anti-LaminA (1:500, sc-20680, Santa Cruz Biotechnology, Inc.), mouse anti-PSD95 (1:500, sc-32290, Santa Cruz Biotechnology, Inc.), mouse anti-synaptophysin (1:1000, sc-17750, Santa Cruz Biotechnology, Inc.), mouse anti-VDAC (1:1000, sc-390996, Santa Cruz Biotechnology, Inc), mouse anti-tau PHF-1 (phosphorylated at Ser396 and Ser394) was a gift by Dr. Peter Davies (Department of Pathology, Albert Einstein College of Medicine, NY, USA), rabbit anti-TOM70 (1:300, PA5-82508, Thermo Fisher Scientific). The fluorescent dyes used were: MitoTracker Red CM-H2Xros (Catalog number: M7513, Thermo Fisher Scientific), MitoTracker Green FM (Catalog number: M7514, Thermo Fisher Scientific), VECTASHIELD Antifade Mounting Medium with DAPI (Catalog number: H1200, Vector Laboratories, Inc).

Ten paraffin coronal sections (5 μm) including the PFC (+2.7 mm from bregma and +3.7 mm from interaural landmark) were stained with 0.5% cresyl violet (n = 6 in each pup). Microphotographs were taken under a 40× objective (Leica, DM6000B, Germany). Nissl-stained cell numbers were counted in a blinded manner using Image J. (NIH, USA). For Immunohistochemistry, the sections were incubated with the following primary antibodies overnight at 4 °C: goat anti-cleaved caspase-3 antibody (L-18) (1:250, Cat.No.sc-1225, Santa Cruz, CA, Germany), mouse anti-Synaptophysin (1:1000, Cat.No.ab8049, Abcam, Cambridge, UK), mouse- anti-PSD-95 (1:1000, Cat.No. MAB1596, Millipore, USA). Signal was detected with Alexafluor 546- or 488-labeled fluorescent secondary antibodies (1:1000, Invitrogen, Carlsbad, CA, USA). Caspase-3 positive cells and PSD-95 and synaptophysin immunofluorescence intensity were evaluated using Image J (NIH, USA).