Therefore, we determined the STC-1 mRNA expression using nested RT-PCR in PB and BM from ESCC patients
treated with radical resection, and their associations with clinicopathological features and 2 year progression-free survival (PFS) were further evaluated. Methods Study population This study enrolled 85 ESCC patients treated with radical resection at Jinling Hospital from July 2006 to July 2008. Patients consisted of 54 males and 31 females, with a median age of 62 (range, 44–83) years. Tumor stage was conducted according to the 7th edition of the TNM staging system of the International Union Against Cancer , and patients were at stages I (n = 18), II (n = 25), III (n = 33) and IV(n = 9, supraclavicular Emricasan purchase or para-aortic click here lymph nodes metastasis). Cellular differentiation was graded according to the WHO grading system. Ethical approval was obtained from the hospital and informed consent was obtained from all patients prior to sample examination. Clinical follow-up data were available for all the patients. For each patient, 10 mL PB before surgery was collected, and PB mononuclear cells were isolated using Lymphocyte separation medium (Sigma, St. Louis, USA) according to the manufacturer’s protocol. Also, 5–10 mL of BM was aspirated from ribs during surgical treatment, and mononuclear cells were isolated from BM by Ficoll gradient centrifugation and
then aliquoted to isolate RNA. PB and BM samples from Rebamipide 40 patients with benign Doramapimod supplier esophageal disease were also collected. Immunohistochemical staining Formalin-fixed, paraffin-embedded samples used for immunohistochemistry were sectioned at 2 μm thickness. Sections were deparaffinized using xylene, dehydrated by gradient ethanol, and then
rehydrated with deionized water. Heat-mediated antigen retrieval was run by autoclave treatment (120°C for 2 min in 1 mmol/L ethylenediaminetetraacetic acid [EDTA], pH of 8.0) and then followed by cooling at room temperature. Incubation with a polyclonal goat anti-STC-1 antibody (diluted 1:200, Santa Cruz Biotechnology, CA, USA) was performed overnight at 4°C. After washing with phosphate-buffered saline (PBS), sections were then incubated with donkey anti-goat secondary antibody (Santa Cruz) for 30 min at room temperature. Coloration was performed with 3,3-diaminobenzidine. Nuclei were counterstained with hematoxylin. PBS was used as a negative control for the staining reactions. Immunostaining results were evaluated independently by 3 pathologists. The percentage of positive cells was rated as follows: 0 score for 0–5%, 1 score for 6–25%, 2 scores for 26–50%, and 3 scores for more than 50%. The staining intensity was rated as follows: 0 score for no staining, 1 score for weak staining, 2 scores for moderate staining, and 3 scores for strong staining .