Rabbit anti vimentin polyclonal antibody

Passage 3 (P3) KSCs were fixed in 4% paraformaldehyde/PBS buffer in a 24-well plate (Corning Inc., USA) at room temperature, permeabilized with 0.1% Triton X-100 (Beijing Chemical Factory), blocked for 30 min in 10% goat serum (BD, USA), and then incubated with primary antibodies at 4°C overnight. The primary antibodies included mouse anti-E-cadherin polyclonal antibody, rabbit anti-ZO-1 polyclonal antibody, rabbit anti-fibronectin polyclonal antibody (Santa Cruz, USA), mouse anti-N-cadherin polyclonal antibody, mouse anti-α-SMA polyclonal antibody (Sigma, USA), and rabbit anti-Vimentin polyclonal antibody (Cell Signaling Technology, USA). The secondary antibodies—tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG, goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG, or goat anti-mouse IgG (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., China)—and 4′-6-diamidino-2-phenylindole (DAPI, BD, USA) were added sequentially. Imaging was performed with an IX70 confocal fluorescence microscope (Olympus, Japan).

Primary cultured cancer-associated fibroblasts derived from human BC were confirmed by immunocytochemistry using a mouse anti-α-SMA monoclonal antibody (1:100; Abcam, USA) and a rabbit anti-Vimentin polyclonal antibody (1:100; Cell Signaling Technology, USA). Briefly, the cells were seeded at a density of 1 × 10⁵ cells/well in 12-well culture plates. After incubation for 24 h at 37 °C and 5% CO₂, the cells were fixed in 4% formalin for 15 min and permeabilized in 0.1% Triton X-100 for 5 minutes. To reduce non-specific staining, the cells were treated with 4% bovine serum albumin in PBS with 0.1% Tween 20 for 30 min. The cells were then incubated with an anti-α-SMA monoclonal antibody and an anti-Vimentin antibody overnight at 4 °C. After three successive rinses in PBS, the cells were incubated for 15 min with avidin-biotin horseradish peroxidase complex, and the reaction was visualized using 0.02% 3,3′-diaminobenzidine tetrahydrochloride as the chromogen in Tris-HCl buffer. Hematoxylin was used to counterstain the nuclei.