0) preheated to 80°C, maintaining this temperature and keeping se

0) preheated to 80°C, maintaining this temperature and keeping sections in this solution for 10 min in a microwave pressure cooker. After allowing the sections to cool to room temperature, the slides were rinsed in PBS (pH 7.4). Endogenous peroxidase activity was BB-94 clinical trial blocked by incubation of the tissue samples for 10 min in 3% hydrogen peroxide. Samples were incubated for 45 min with the primary antibodies at room temperature in a moisture chamber. VEGF determination and analysis Samples were incubated with mouse anti-VEGF monoclonal antibody (1:100) Necrostatin-1 datasheet (Abcam, Cambridge

MA, USA) in BSA 1% in PBS for 45 min. After washing with PBS, binding of the primary antibodies was revealed by incubation for 20 min with LSAB+ System Link (DAKO, Carpinteria, CA, USA) and LSAB+ HRP, (Streptavidin HRP kit, DAKO). The slides were rinsed with PBS and exposed to diaminobenzidine for 5 min. After washing with PBS and counter-staining with hematoxylin, the slides were dehydrated by graduated alcohols and xylol, and mounted with Poly-mount. Numerical proportions of stained cells were established by analyzing 10 high-power fields (400×) in each section.

Only cytoplasmic staining was considered positive. Intensity was graded on a semi-quantitative scale from 0–3. Graduation of expression was considered negative if fewer than 5% of cells were stained. Determination of vascular density The samples were incubated for 45 min with mouse anti-CD34 monoclonal antibody (1:200) Thiamet G (Biocare Medical, Concord, CA, USA) as a marker for vascular endothelial cells. Three separated, highly vascularized areas (“”hot spots”"), PRI-724 previously identified in high-power fields (100×, then 400×), were analyzed by two pathologists by means of optic microscopy without previous knowledge of hCG determinations. Any immunostained vessel clearly separated from adjacent vessels with no muscular wall and within the optic field was considered a neovascularization vessel.

Vascular density (VD) was considered as the average of the three evaluated zones. Statistical analysis For descriptive purposes, continuous variables were summarized as arithmetic means, medians, and standard deviations (SDs), while categorical variables were expressed as proportions and confidence intervals (CIs). Inferential comparisons were carried out using the Student t or the Mann-Whitney U test, according to data distribution determined by the Kolmogorov-Smirnov test. Chi square or Fisher exact test was used to assess significance between categorical variables. Statistically significant and borderline-significant variables (p < 0.1) were included in the multivariate logistic regression analysis. Overall survival time was measured from day of surgery to date of death or last follow-up visit and analyzed with the Kaplan-Meier method, and comparisons among sub-groups were performed with the log-rank test. For survival curve analysis, all variables were dichotomized.

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