Pierce ecl plus western blotting substrate detection kit

The frozen hippocampal tissues of AD models and controls were homogenized by sonication and centrifuged at 15,000 × g. Protein was collected from the supernatants and measured by BCA protein assay kit (Thermo Fisher Scientific). 50 μg protein was separated with 15% sodium dodecyl sulfate-PAGE gels and then electrotransferred onto polyvinylidene fluoride membranes (Millipore, Shanghai Trading Company Ltd., Shanghai, China). Separated protein was then immunoblotted with mouse anti-GFAP (1:400; Wuhan Servicebio Technology Co., Ltd., Wuhan, China) and mouse anti-GAPDH (1:5000; Wuhan Servicebio Technology Co., Ltd., Wuhan, China). The membranes further reacted with anti-mouse IgG (1:20,000; Thermo Fisher Scientific). Immunoblot signaling was visualized with the Pierce ECL-Plus Western Blotting Substrate detection kit (Thermo Fisher Scientific), followed by X-ray film exposure and image capture in a laser scanner. Quantitative analysis of GFAP-positive dots was performed with Image-Pro Plus software.

Proteins were extracted from purified BMMCs using RIPA Buffer (Xianfeng Biotech, Xi’an, China) containing protease and phosphatase inhibitors according to the manufacturer’s instructions. Equal amounts of protein lysates from each group were separated by SDS-PAGE and transferred to a methanol-activated PVDF membrane (Millipore, Beijing, China). The membrane was blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk for 2 h, and then incubated with primary antibody at 4 °C for 24 h, washed, and incubated with secondary goat anti-rabbit antibody conjugated with HRP (Abgent, San Diego, USA). Western blot analysis was conducted according to standard procedures using a Pierce™ ECL Plus Western Blotting Substrate detection kit (Thermo Fisher Scientific, Rockford, USA). Image capture and analysis were performed using a GE Image Scanner (GE Healthcare, USA). Protein levels were normalized to those of β-actin. Primary occludin (ab31721) antibody was from Cell Signaling Technology (Cambridge, MA, USA), and primary β-actin (66009-1) antibody was from Proteintech (Chicago, USA).