Bm3895

GC tissue specimens were cut into 5-μm-thick sections. The sections were blocked with normal goat serum and immunostained with primary antibodies to active YAP, CDX2 and Rab27a overnight at 4 ºC, and then with biotin-labeled secondary antibody goat anti-rabbit or goat anti-mouse (BM3894 or BM3895, 1: 500, Boster). Next, the sections were incubated with 50 μL streptomyces anti-biotin–peroxidase solution at room temperature for 10 min and developed with DAB. Following counterstaining, dehydration, clearing and mounting, the sections were observed with a microscope. Primary antibody was substituted by PBS. Positive cells were brown-yellow and the sum of the integrated optical density (IOD) was analyzed using Image-ProPlus6.0 [20 (link)].

We prepared the samples from the xenograft tumor mice using formalin-fixed, paraffin-embedded samples. Paraffin-embedded samples were cut into 4 μm thick sections, which were then blocked with 3% hydrogen peroxide for 60 min at room temperature. After antigen retrieval, the sections were incubated with antibodies against PI3K (EM1701-62, HUABIO, 1: 200) and Ki-67 (9449 s, Cell Signaling Technology, 1: 500). After incubation with the primary antibodies, the tissue sections were incubated with the appropriate secondary antibodies (BM3895, BOSTER Biological Technology, 1: 1000) for 1 h at room temperature and then stained with diaminobenzidine and hematoxylin.

After all the behavioral tests were finished, the animals were anesthetized with pentobarbital sodium (50 mg/kg) before being decapitated for the brain. The hippocampi and the cortexes in the two hemispheres of the brain were separated on ice and then used for ELISA detection and the Western Blot experiment, respectively. The levels of tau (ab210703, 1 : 1000), p-tau (ab210703, 1 : 1000), GFAP (ab7260, 1 : 5000), Iba-1 (ab178846, 1 : 1000), and ß-actin (ab6276, 1 : 5000) (all the antibodies were from Abcam company) in the hippocampus and the cortex were determined, respectively. The secondary antibodies were from Boster (bm3895 and bm3894). The homogenates from hippocampus and cortex samples of mice were separately added along with 30 mg total DNA to 10% SDS-PAGE gel for separation. They then were electrophorized under constant voltage, transferred onto a membrane, and incubated with specific antibodies for immunoblotting assays using the ECL immunoblotting analysis system. Each gel contains molecular weight markers (10–170 kDa). The band data obtained were processed with ImageJ (NIH) and the background mean and its standard error were calculated.