Rabbit anti vimentin

The tumor tissues were evaluated for cell proliferation using Rabbit anti-human Ki-67 monoclonal antibody (Maixin Biotech, Fuzhou, China). Rabbit Anti-P-CK/Cytokeratin AE1 + AE3 (Bioss, Beijing, China) (CK) and Rabbit Anti-Vimentin (Bioss, Beijing, China) were used to determine the origin of cells in the tumor tissue, CK-positive epithelial cells, or Vimentin-positive stromal cells. Formalin-fixed, paraffin-embedded tissue blocks were cut into 5-μm sections and mounted on positively charged slides. Tissue sections were deparaffinized with xylene and rehydrated with graded alcohol solutions; they were then incubated in citrate buffer (pH 6.0) at 210°C for 10 min and at 160°C for 10 min in a pressure cooker. After incubation in 3.0% hydrogen peroxide for 10 min and PBS wash, the tissue sections were immersed in working solution of anti-Ki-67, anti-cytokeratin, or anti-Vimentin for 1 h at 37°C. Tissue sections were exposed with a second antibody (MaxVision™ HRP-Polymer anti-rabbit IHC kit, Maixin Bio, Fuzhou, China) for 15 min at room temperature. Finally, the sections were incubated in DAB chromogen and then counterstained with hematoxylin. Positive and negative controls were used throughout all immunostaining protocols. The ratio of Ki-67-positive cells was defined as the percentage of positive cancer cells by counting 2000 cancer cells at ×200 microscopically.