【文獻標題】Upregulation of microRNA-141 contributes to suppressing epithelial-mesenchymal transition and lymph node metastasis in laryngeal cancer through HOXC6-dependent TGF-βsignaling pathway
ImmunohistochemistryThe positive expression rates of HOXC6 in laryngeal cancer and adjacent normal tissues were detected by immunohistochemistry. Laryngeal cancer tissues and adjacent normal tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated using 80%, 90% and 100% ethanol successively, cleared with n-butanol, placed in a paraffin box at 60°C and embedded in paraffin. After that, the tissues were cut into 5 μm-thick slices, deparaffinized with xylene after being heated at 60°C for 1 h. The slices were then dehydrated by gradient alcohol and soaked in 3% H2O2 for 10 min, followed by antigen retrieval. Next, the slices were boiled in 0.01 M citric acid buffer (pH = 6.0) at 95°C for 15 - 20 min, and incubated with 100 μL 5% bovine serum albumin (BSA) blocking solution at 37°C for 30 min, followed by incubating with 100 μL primary rabbit antibody to HOXC6 (1 : 50, PB0948, Wuhan Boster Biological Technology, Ltd., Wuhan, Hubei, China) overnight at 4°C. The slices were then incubated with biotin-labeled goat anti-rabbit (1 : 100, HY90046, Shanghai HengYuan Biological Technology Co., Ltd., Shanghai, China) at 37°C for 30 min and with streptavidin-peroxidase solution (Beijing Zhongshan Biological Technology Co., Ltd., Beijing, China) at 37°C for 30 min, and then developed by diaminobenzidine staining solution (Beijing Bioss Biological Technology Co., Ltd., Beijing, China) at room temperature. Subsequently, the slices were soaked in hematoxylin for 5 min, rinsed with 1% hydrochloric acid for 4 s and washed under tap water for 20 min. Brownish-yellow cells were confirmed as HOXC6 positive cells [23]. Five high-magnification visual fields were randomly selected from each slice with 100 cells per field. HOXC6 positive expression was defined by the percentage of positive cells: number of HOXC6 positive tumor cells/number of total tumor cells > 10% was counted as positive (+), < 10% was regarded as negative (-). Experiments were repeated 3 times independently.
