Proteins with score value over 60 were positively identified. Western blotting Protein samples were separated by 10%

SDS-PAGE gels and then transferred to PVDF membranes. After blocked with 5% defatting milk for 1 h at 37°C, the membranes were MM-102 price incubated with anti-vimentin (1:1000, Thermo Scientific, USA) at 4°C overnight. After washing with 0.5% PBS-T (PBS with Tween-20) for three times, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. Membranes were washed again with PBS-T. The signals were detected by using the Western blotting chemiluminescent kit (Pierce), quantified by densitometry and analyzed by using Quantity One image analysis system (BioRad). The detection of β-actin (1:5000, Santa Cruz, USA) was used as the inner control. Patients Paraffin-embedded check details melanoma specimens (n = 70) were obtained from the Tianjin Cancer Hospital from 1998 to 2003. Detailed pathological and clinical data were collected and none of the patients had received treatment before

operation. Clinical outcome was followed from the date of surgery to the date of death or until Jan, 2009. The summary of the clinicopathological data of the cases is shown in Table 1. Of 70 enrolled cases, 43 males and 27 females (mean age, 54.96 ± 12.60 years). The sites of melanomas were trunk (13/70), limbs (27/70), head and neck (13/70), digestive system (8/70) and genital system (9/70) respectively. We categorized them into two groups: cutaneous melanoma (53/70) and extra-cutaneous melanoma (17/70). The survival durations ranged from 1 to 113 months (mean, 34.90 ± 27.42 Dolutegravir cell line months). Primary melanoma with hematogenous metastasis BVD-523 molecular weight was observed in 29 cases. This study was approved by the ethics committee. Table 1 Correlation of vimentin expression with clinicopathologic features of 70 primary melanoma patients Patients Characteristics

Factors n vimentin expression χ2 p value (N = 70)     low high     Age(y) < 60 43 21 22 0.128 0.808   ≥60 27 12 15     Gender Male 43 15 12 1.248 0.328   Female 27 18 25     Location Cutaneous 45 22 23 0.154 0.804   Extra-cutaneous 25 11 14     TNM Stage I 16 10 6 2.145 0.342   II 24 11 13       III 30 12 18     Lymph node metastasis positive 28 12 16 0.344 0.629   negative 42 21 21     Hematogenous metastasis positive 29 8 21 7.599 0.008*   negative 41 25 16     Immunohistochemical staining of patients samples Seventy formalin-fixed, paraffin-embedded melanoma patients samples were cut into 4 μm sections and dried overnight at 65°C. The sections were deparaffinized in xylene and rehydrated through graded alcohols into water. Endogenous peroxidase was blocked with 3% hydrogen peroxid for 20 min in the dark chamber. Microwave antigen retrieval was performed using citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100°C in a microwave oven. After rinsing with PBS, the slides were incubated with anti-vimentin (1:100, Thermo Scientific, USA) overnight at 4°C.