Anti muc 1

Colon tissues were collected at endpoint and embedded in paraffin; then, colon tissues go through deparaffinating, antigen retrieval, and blocked with serum. After blocking, colon samples were incubated with anti-MUC-1 (Servicebio, Wuhan, Hubei, China) at 4°C overnight. The second day, colon tissues were incubated with horseradish peroxidase (HRP)-labeled secondary antibody at 37°C for 1h. Next, several steps were conducted, including SABC reaction, DAB chromogenic, and tissue dehydration. The staining morphology was photographed using microscope (Leica). The H-score of intensity was analyzed by Aipathwell software (Servicebio).

IEC-6 cells were lysed by RIPA lysis buffer (Meilunbio, Dalian, Liaoning, China). SDS loading buffer was added to the sample and incubated at 100°C for 10 min. Sample proteins were electrophoretically separated by 30% SDS-PAGE gels (Servicebio) and transferred to a 0.45µm polyvinylidene difluoride (PVDF) membrane. The proteins were blocked with 5% skim milk in Tris-buffered saline. Then samples were incubated with primary anti-MUC-1 (Servicebio) and anti-β-actin (Abclonal, Wuhan, Hubei, China) at 4°C overnight. Next, the samples were incubated with HRP-labeled goat anti-rabbit IgG H&L (Servicebio). Sample bands were visualized using enhanced chemiluminescence (ECL) and analyzed with the ProteinSimple instrument (Bio-Techne, Minnesota, USA).