Blue x ray film

To confirm the protein level of phosphorylated tau, we performed the western blot as previously described [24 (link)]. Briefly, the frozen cortex was homogenized using radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCL, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris, pH 8.0) containing protease inhibitors (Roche Applied Science, Mannheim, Germany) and cocktail of phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA) and centrifuged at 13,000 rpm for 10 min at 4 °C. After lysate samples were quantified using Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), loaded onto an 10% SDS-PAGE and the proteins were transferred onto a PVDF membrane. The membrane was blocked the 3% BSA in TBS-T for 1h at room temperature and then incubated with appropriate antibody for overnight at 4 °C. After three times wash in TBS-T, the membrane incubated with secondary antibody for 1 h. The protein band was detected using ECL (Millipore, Burlington, MA, USA) and BLUE X-ray film (AGFA, Mortsel, Belgium). The band quantification was performed using the Image J software v1.4.3.67.

Cytokines, chemokines and angiogenic factors using commercially available protein array systems (Proteome profiler^TM Arrays, R&D Systems) according to the manufacturer’s protocol. CRT-MG cells were either untreated or treated with necrotic cells for 24 h, and then supernatants were collected and subjected to protein array. Sample and reconstituted detection antibody mixture were incubated at RT for 1 h and the prepared mixtures were added on each membrane at 4 °C for overnight. And diluted streptavidin-HRP treated into each membrane for 30 min at RT on a rocking platform shaker. Membranes were exposed to blue X-ray film (AGFA, Mortsel, Belgium) by using chemiluminescent reagent for 3–10 min. The changed spot intensities were measured by the spot intensity divided by reference spot intensity, which using Image analysis software.