Anti snail2

Cryosections with a thickness of 4 μm were prepared using a cryostat and were fixed in 4% paraformaldehyde for 15 min. After blocking, the cryosections were incubated with primary antibodies and then with a fluorescein Cy3-FITC-labelled secondary antibody (1:100; Proteintech, Wuhan, China). Fluorescence images were recorded using a TCS SP5II confocal microscope (Leica, Bensheim, Germany). The following primary antibodies were used: anti-desmin (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-podocalyxin (1:100; R&D Systems, Minneapolis, MN, USA), and anti-snail2 (1:100, Proteintech). Podocytes were seeded onto clean glass coverslips, fixed with 4% paraformaldehyde, and permeabilised with 0.2% Triton X-100. The slides were incubated with an anti-PCNA (1:100, Proteintech), anti-synaptopodin (1:100; Proteintech), anti-CDK4 (1:100; Abcam, Cambridge, UK), anti-P-YAP (1:100; Cell Signaling Technology, Danfoss, MA, USA), or anti-snail2 (1:100, Proteintech) antibody.
For immunohistochemistry analysis, after deparaffinisation, rehydration, antigen retrieval, and blocking, the sections were incubated with an anti-PCNA (1:100), anti-CDK4 (1:100), anti-desmin (1:100), anti-YAP (1:100, Proteintech), or anti-cyclin D1 (1:100, Cell Signaling Technology) primary antibody and then with a horseradish peroxidase-labelled secondary antibody (Beyotime, Shanghai, China).

Multiplex staining of CK19, IGFBP4 and Slug co-expression on cancer tissues was performed using 4-color kit (WiSee Biotechnology), according to manufacturer's instruction. Three primary antibodies are anti-Cytokeratin 19 (Cat: ab52625, diluted 1:400, Abcam), anti-IGFBP4 (Cat: 18500-1-AP, diluted 1:500, proteintech) and anti-SNAIL2 (Cat: 12129-1-AP, diluted 1:400, proteintech). After applied different primary antibodies (anti-Cytokeratin 19, anti-IGFBP4 and anti-SNAIL2, sequentially), the secondary antibody (HRP conjugated) was added and incubated, followed by tyramide signal amplification (Cat: M-D110051, WiSee Biotechnology). After all antigens being labeled with different antibodies, DAPI (Cat: D1306, ThermoFisher) was used for nuclei staining. Pannoramic MIDI imaging system (3D HISTECH) was then used for scanning the stained slides. The number of target cells were analyzed by HALO software (Indica Labs).

Western blot was performed as previously described [24 (link)]. The primary antibodies used were: anti-CHRNB2 (Abcam, ab41174) for Fig. 5, anti-CHRNB2 (Santa Cruz, sc-58596) for other figures, anti-N-Cadherin (Proteintech, 22018–1-AP), anti-Vimentin (Proteintech, 10366–1-AP), anti-β-catenin (CST, 8480), anti-Cyclin D1 (Abcam, AB16663), anti-CD44 (Proteintech, 15675–1-AP), anti-C-Myc (CST, 5605), anti-SNAIL1(Proteintech, 13099–1-AP), anti-SNAIL2 (Proteintech, 12129–1-AP), anti-Vinculin (Proteintech, 66305–1-Ig) and anti-GAPDH (Proteintech, 10494–1-AP). Quantitative analysis of Western Blot was performed by ImageJ software. Fold changes under individual blots represented the ratio to relevant control (numbers in italic font).