Ab134119

WES, an automated capillary-based electrophoresis system (ProteinSimple, San Jose, CA, USA) was used to analyze protein levels, following the manufacturer’s instructions. Briefly, cell lysates were prepared using the above-mentioned methods and diluted with the 0.1× sample buffer to a protein concentration of 1 mg/mL. Then, the lysates were mixed with the 5× fluorescent master mix in a 4:1 ratio, and the mixtures were heated at 95 °C for 5 min. The prepared lysates, antibody dilution buffer, primary antibody, anti-mucin 2 (MUC2) antibody (ab134119, 1:50, Abcam), HRP-conjugated secondary rabbit antibody, and chemiluminescent substrate mix (luminol:peroxide mixture in a 1:1 ratio) were dispensed into predetermined wells in an assay plate. For the total protein assay, the total protein label reagent and total protein streptavidin-HRP were used instead of the primary antibody and the HRP-conjugated secondary antibody. After centrifugation, a wash buffer was added to the plate. The plate and capillary cartridge were placed into a WES instrument (ProteinSimple), and the runs were started. After the analysis, the resulting data were evaluated using the Compass software v3.1.7 (ProteinSimple). MUC2 levels were normalized to that of the total protein content.

Before staining, antibody was optimized and antigen was retrieved. The sections were deparaffinized and rehydrated. With the condition of citrate buffer pH 6 pretreatment, monoclonal primary antibody against MUC2 (EPR6145, ab134119, Abcam diluted 1:1000) was applied for the immunohistochemical staining. After phosphate-buffered saline (PBS) washing, sections were incubated with secondary antibody coupled to EnVision/HRP (zhongshan goldenbridge biotechnology CO). All of the results were visualized by Image-Pro Plus 6.0 view software (Media Cybernetics, America).