Hpa001893

Frozen sections were fixed with 100% ethanol for 15 minutes and permeabilized using 0.05% Tween20 twice (2 minutes per treatment). Blocking was performed using 3% bovine serum albumin for 30 minutes at RT. After washing with phosphate belanced solution (PBS), sections were incubated with rabbit anti-human SIX1 primary antibody (1:100 dilution) (HPA001893, Sigma, St. Louis, MO, USA), rabbit anti-human EYA1 primary antibody (1:100 dilution) (ab85009, Abcam, Cambridge, MA, USA), and mouse anti-human HSA primary antibody (1:1,000 dilution) (MAB1455, R&D Systems, Minneapolis MN, USA) or mouse anti-human GFAP primary antibody (1:100 dilution) (MA5-12023, ThermoFisher, Waltham, MA, USA) at 4 °C overnight. Sections were then incubated with Alexa Fluor^®488 donkey anti-mouse immunoglobulin G (IgG) and Alexa Fluor^®568 donkey anti-rabbit IgG (1:1,000 dilution) (A21202, A10042, ThermoFisher, Waltham, MA, USA) for one hour at RT. After washing with PBS, sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Finally, coverslips were mounted with anti-fade mounting medium, and immunofluorescent signals were visualized and recorded using an Olympus DP72 microscope and the cellSens Standard 1.5 software. (Olympus Corporation, Tokyo, Japan)

CRC cancerous tissues and their adjacent non-cancerous tissues and cultured CRC cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (ThermoFisher Scientific, #89901) mixed with protease inhibitor (ThermoFisher Scientific, #78425). Equal amounts of total cell extracts were resolved in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. After transforming onto polyvinylidene fluoride membranes and blocking with 5% milk for 1 h, proteins were incubated with primary antibodies including anti-SIX1 (Sigma-Aldrich, #HPA001893), anti-EYA1 (Sigma-Aldrich, #HPA028917), anti-CCNA1 (Sigma-Aldrich, #SAB1409961), anti-TGF-β (Abcam, Cambridge, MA, USA, #ab92486), and anti-glyceraldehyde 3-phosphate dehydrogenase (Abcam, #ab8245) overnight at 4°C, respectively. The membranes were further incubated with secondary antibodies at room temperature for 1 h and then protein bands were visualized using an enhanced chemiluminescence kit (ThermoFisher Scientific, #32106).