Anti fading fluorescence media

Immunocytochemistry was performed on mouse brain cryosections or PFA fixed cells according to standard methods [25 ]. Briefly, sections or cells were blocked in PBS containing 0.3% Triton X-100 and 10% donkey serum for 1 h in room temperature. They were then incubated in the same solution with primary antibodies overnight at 4 °C and immersed in fluorescent secondary antibodies for 1 h at room temperature. After counterstaining with DAPI for 5 min, all sections were mounted with anti-fading fluorescence media (Vector Laboratories) under a glass coverslip. All coverslips were mounted on glass slides. Experiments were the results of triplicate cultures established on separate days.

Apoptotic and necrotic events in cell culture were assayed by annexin V/propidium iodide (V13245, Life Technologies) following the manufacturer’s protocol. In brief, coverslips were washed with cold PBS and immediately incubated with working solution containing propidium iodide and Alexa Fluor® 488 annexin V diluted in an annexin-binding buffer for 15 min. After washing with annexin-binding buffer, coverslips were mounted with anti-fading fluorescence media from Vector Laboratories (Burlingame, CA, USA).