Rabbit anti snail

Immunofluorescence analyses were performed as described previously^{12 (link)}. Paraffin sections of iERM tissues were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). Sections were incubated with the following primary antibodies: rabbit anti-SNAIL (1:100, Proteintech, Rosemont, IL), mouse anti-TGF-β1 (1:50), mouse anti-S100 (1:50, Thermo Fisher Scientific), goat anti-TβRII (1:50), mouse anti-α-SMA (1:200, R&D Systems), goat anti-SM22 (1:100, Abcam, Cambridge, UK), mouse anti-GS (1:100, Millipore), and mouse anti-GFAP (1:100, Leica, Exton, PA) antibodies. Secondary antibodies for fluorescent detection were AlexaFluor 488 and 546 (1:400, Thermo Fisher Scientific). Nuclei were counterstained with DAPI, and sections were examined using a Biorevo BZ-9000 microscope (Keyence).

Western blot analyses were performed on cell lysate prepared from three esophageal cancer cell lines and esophageal tissue as described previously. Equal amount of protein lysate were loaded and separated on 10 % SDS–polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore, USA). After being blocked by 5 % fat-free milk in TBS buffer, membranes were probed with the following antibodies overnight at 4 °C: mouse monoclonal anti-PFN2 (Abcam; 1:1000 dilution), rabbit anti-E-cadherin (Santa Cruz; 1:200 dilution), rabbit anti-Vimentin (Abcam; 1:1000 dilution), rabbit anti-Snail (Proteintech; 1:500 dilution), rabbit anti-Slug (Proteintech; 1:200 dilution), rabbit anti-ZEB1 (Proteintech; 1:500 dilution) and mouse anti-β-actin (Zhongshan Biotechnology; 1:1000). Bound antibodies were detected with secondary HRP-conjugated antibodies (Santa Cruz) for 2 h at room temperature. After washing, the resulting bands were visualized using the standard ECL procedure (Kangwei, Beijing). Images were acquired using the image acquisition system (BioRad, USA) and their grayscale value was analyzed by the image analysis program (Gel-Pro Analyzer 4.0, USA).