Rabbit anti beclin1

Mouse anti-ROCK1 antibody (Abcam, 1:500 dilution), rabbit anti-Beclin1 (Novus, 1:10,000), rabbit anti-APP-CTF (Sigma-Aldrich, 1:5000), anti-mouse sAPPα (IBL, 1:50), anti-mouse myc (Abcam, 1:1000), anti-rabbit LC3 (Cell Signaling, 1:100) and mouse anti-β-actin (Sigma-Aldrich, 1:1000) were used. Loading controls (β-actin) were used for Western blot standardization. Lipofectamine 2000 was sourced from Invitrogen. 3-methyladenine (3-MA) was purchased from Sigma Aldrich (St. Louis, MO, USA) and 3-MA was dissolved in double distilled water and titrated to 1 mM.

APP/PS1 mice were provided by Model Animal Research Center of Nanjing University. APP/PS1 mice have been intensively characterized for Aβ plaques load and AD behavioral phenotypes. In this experiment, 10-month-old APP/PS1 transgenic mice and age-matched C57BL/6J were used. Mice were deeply anesthetized and perfused with 10% formalin, and their brains were removed and fixed in 4% paraformaldehyde in PBS overnight at 4°C. Whole brains were cryoprotected in 25% sucrose in 0.1M PBS at 4°C until sectioning. The brain tissues were cut in to coronal 30 μm sections. Immunohistochemistry (IHC) for APP and Beclin1 were performed using primary antibodies of 6E10 (1:100, Novus) and rabbit anti-Beclin1 (1:100, Novus) respectively (Sugrue et al., 2010 (link)). APP and Beclin1 immunopositive neurons were quantified according to the previously established method (Dlugos and Pentney, 2002 (link)).