Anti e cad

Cell immunofluorescence analysis was performed as described by previous studies (18 (link)). Briefly, the SW620 cells were fixed in 4% paraformaldehyde then permeabilized for 20 min using PBS containing 0.5% Triton X-100. Thereafter, the cells were blocked with 3% BSA containing 0.025% Triton X-100 and 5% FBS (30 min, at room temperature). The cells were then incubated overnight with primary antibodies (anti-E-cad and anti-N-cad, Abcam, Cambridge, UK) at 4°C or using the EdU Proliferation Kit (20 μM EdU for 3 hours, Abcam). After washing three times, the cells were incubated with the appropriate secondary antibodies. They were then washed, after which 4,6-diamidino-2-phenylindole (DAPI; Invitrogen) was used for nuclear counterstaining, for 5 min. Finally, immunofluorescence was visualized using a confocal microscope (LSM710, Carl Zeiss, Shanghai, China).

Antibodies to histatin-1, E-cad, lactoferrin and ASMA were used for immunofluorescence staining of MMCR tissue sections. Xylene and rehydration with serial ethanol dilutions were used for deparaffinization. Slides were washed twice for 5 minutes in 0.25% Triton X-100 for permeabilization and blocked for 2 hours at room temperature with 2% BSA and 10% normal donkey serum in PBS. Slides were incubated overnight at 4°C with the primary antibody diluted in blocking solution (1:100) (Anti-Lactoferrin; Cat. #ab6410-100; Abcam, MA, USA, Anti-Histatin-1; Cat. #ab81089; Abcam, MA, USA, Anti-E-cad; Cat. #ab76055; Abcam, MA, USA and Anti-ASMA; Cat. #MS-113-P0; Thermo Scientific, MA, USA). The next day, the slides were washed twice for 5 minutes in PBS and incubated for 1–2 hours with respective secondary antibodies (Jackson ImmunoResearch laboratories INC., PA, USA) diluted in blocking solution (1:200–800). Vecta shield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Cat. #H-1200; Vector Labs, CA, USA) was placed over the slides and covered with a glass coverslip. Slides were analyzed using the Zeiss LSM 710 Confocal Microscope.
Similarly, cultured human MLG derived epithelial cells were cultured on the chamber slides and fixed with 4% paraformaldehyde for immunostaining with antibodies to histatin-1, lactoferrin, E-cad, and ASMA.

Protein preparation and Western blot assay were performed as described previously [31 (link)]. The antibodies used were as follows: anti-TRAIP (ProteinTech, dilution at 1:1000), anti-β-actin (ProteinTech, dilution at 1:4000), anti-RB, anti-P-RB, anti-E2F1, anti-Cyclin D1, anti-Cyclin E1, anti-P21, anti-MMP9, anti-Twist, anti-Slug, anti-E-cad (abcam, all at dilution 1:1000), anti-MMP2, and anti-Vimentin (abcam, all at dilution at 1:500).

Primary HSCs from mice were immobilized in 4% paraformaldehyde. After fixation, cells were washed with PBS and blocked with 5% BSA. Then, cells were incubated with anti-E-cad (1:100 dilution; Abcam) and anti-desmin (1:100 dilution; Abcam) antibodies overnight at 4 °C. Then, cells were stained with fluorescence-labeled anti-rabbit Alexa 594 (1:50 dilution; Dianova)-conjugated antibodies. For nuclear counterstaining, 4,6-diamidino-2-phenylindole (DAPI) was applied.