Vegf c

Cells were harvest with RIPA lysis solution (Beyotime, China). The total protein concentration was quantified by the BCA method (Thermo Fisher Scientific, Inc.). Western blotting (immunoblotting) was conducted in accordance with a standard experimental procedure. Total protein was separated by 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat dry milk in TBST (40 mM Tris–HCl, 0.5 M NaCl, 0.1 Tween-20, pH 7.4) at room temperature for 2 h. Primary antibodies (SARG, Proteintech; VEGF-C, Proteintech; VEGFR-3, ABACM; ERK1/2, Proteintech; Phosphorylated-p44/42 ERK1/2, Cell Signaling Technology; GAPDH, Santa Cruz Biotechnology) were diluted in the antibody diluent at 4°C overnight, and secondary antibodies (anti-rabbit 1:10000 and anti-mouse 1:10000) were diluted in TBST for 1 h at room temperature. The detection and quantification of protein bands were captured with the Image Quant LAS 4000 Mini Imager (GE Healthcare Bio-Sciences).

Multiplexed immunofluorescence (mIF) was performed by staining 4‐μm‐thick formalin‐fixed, paraffin‐embedded whole tissue sections with standard, primary antibodies sequentially and paired with a TSA 7‐color kit (D110071‐50T, Yuanxi Bio). The primary antibodies were MUC1 (Abcam; catalog no. ab109185; RRID:AB_10862483), THBS1 (Abcam; catalog no. ab267388), FGF7 (Sabbiotech; catalog no. 31162‐1), MS4A4A (Abcam; catalog no. ab271069), ZEB1 (Proteintech; catalog no. 21544‐1‐AP; RRID:AB_10734325), VEGFC (Proteintech; catalog no. 22601‐1‐AP; RRID:AB_2879132), and CD68 (Abcam; catalog no. ab213363; RRID:AB_2801637). After multiplexed immunofluorescence staining, each slide was treated with two drops of DAPI, washed in distilled water, and manually coverslipped. Slides were air‐dried and mounted with anti‐fade mounting medium, and pictures were taken with PANNORAMIC MIDI II. Images were analyzed using Indica Halo software.

Thyroid samples of patients were collected by the Taizhou Central Hospital. Immunohistochemistry was performed as follows. Sample sections were dewaxed with xylene, hydrated with ethanol, then run through an antigen repair protocol. Endogenous peroxidases were neutralized by 0.3% H₂O₂ in dH₂O for 10 min. After washing, slides were blocked with 10% ready-to-use goat serum for 0.5 h at room temperature. Tissue sections were incubated with the primary antibodies(SARG, Proteintech; VEGF-C, Proteintech; VEGFR3, Proteintech)overnight. After washing with PBS, samples were incubated with HRP anti-rabbit IgG (1:200) at room temperature for 30 min and diaminobenzidine (DAB) was used as a sensitive chromogen and then counterstained with hematoxylin. Slides were analyzed by taking photos with a microscope (BX53, Olympus, Japan).