Ab251662

After treatment, the cells were scraped off, transferred into a 2.5 ml EP tube and added with 150 µl mixture of radioimmunoprecipitation assay (RIPA) and protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA), followed by ultrasonic dispersion using an ultrasonic apparatus (40 A, 3 sec/time, repeated 3 times). After centrifugation at 12,000 × g at 4°C for 15 min, the supernatant was taken as the tissue protein. The protein concentration was measured using the bicinchoninic acid (BCA) kit (Beyotime Institute of Biotechnoogy). After denaturation, the total protein was separated using 10% acrylamide gel, transferred onto a 0.22 µm nitrocellulose membrane (EMD Millipore) for 1.5 h, sealed with 5% skim milk for 1 h and incubated with rabbit anti-human fibulin-2, β-catenin, cyclin D1, C-myc and GAPDH polyclonal antibodies (cat. nos. ab251662, ab16051, ab226977, ab39688 and ab9485, respectively; Abcam; diluted at 1:1,000) overnight. Then the band was incubated with the goat anti-rabbit IgG secondary polyclonal antibody (cat. no. ab6721; Abcam; diluted at 1:300) for 1 h, and the target protein band was developed using the ECL system (Bio-Rad Laboratories). The relative content of the target protein was calculated as: gray value (target protein)/gray value (corresponding internal reference band).

After 49 cases of gastric cancer and para-carcinoma tissue specimens were fixed with 4% formaldehyde, routinely dehydrated and embedded into paraffin, the paraffin-embedded tissues were sliced into 4 µm sections, followed by IHC using streptavidin-peroxidase (SP) staining: After dewaxing with xylene and dehydration with gradient ethanol, antigen retrieval was performed using sodium citrate buffer via microwave, and the peroxidase was blocked with 3% H₂O₂ blocker. The sections were sealed with 10% donkey serum, phosphate-buffered saline (PBS) was added as the negative control, and the primary rabbit anti-human fibulin-2 and β-catenin polyclonal antibodies (cat. nos. ab251662 and ab16051, respectively; Abcam; diluted at 1:200) were added dropwise, followed by incubation in a wet box at 4°C overnight. The next day, sections were washed with PBS 3 times, and incubated with the secondary goat anti-rabbit polyclonal antibody (cat. no. ab6721; Abcam; diluted at 1:200), followed by color development via diaminobenzidine (DAB) and photography under a microscope. The brown and dark brown nuclei under the microscope indicated positive cells, and the number of positive cells was counted. The number of positive cells/total number of cells in the visual field >5% indicated positive expression.