The clinical and pathological data collected included gender, age, hepatitis B surface antigen (HBsAg) status, serum

alpha-fetoprotein (AFP) level, tumor number, tumor size, degree of tumor differentiation, Child-Pugh class, Barcelona Clinic Liver Cancer (BCLC) stage, presence of cirrhosis, ascites, tumor thrombus, and extrahepatic metastasis. The PFS and OS were defined as the time from initiation of sorafenib therapy to the time of disease progression detected by computed tomography or magnetic resonance imaging, or death, respectively. Immunohistochemical staining Expression of VEGFR-2, PDGFR-β, and c-Met were determined by two-step PV-6000 click here immunohistochemistry staining. Specimen slices were dewaxed, rinsed in phosphate-buffered saline (PBS). Antigen retrieval was performed by placing the slides in a high pressure cooker in 0.01 mmol/L citrate buffer, pH 6.0, for 3 minutes at 100°C, followed by cooling for 20 min at room temperature,

rinsing in PBS, treating with 3% hydrogen peroxide in deionized water for 10 min to block endogenous peroxidase, and rinsing again in PBS. Specimens were then incubated at 37°C for 1 hour with primary antibody against VEGFR-2 (dilution ratio 1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA), PDGFR-β (dilution ratio 1:40; Santa Cruz Biotechnology Inc., CA), and c-Met (rabbit anti-human c-Met monoclonal antibody working solution; Epitomics, California, US), followed by rinsing three times in PBS for 2 min each time. Specimens were incubated at 37°C for 20 min with universal IgG antibody-HRP polymer (Zhongshan Jinqiao Co., Beijing, China), and rinsed three times in PBS Small molecule library cost for 2 min each time. Specimens

were placed in DAB solution for color development, rinsed with distilled water, stained again, dehydrated, and sealed with transparent strips. Primary antibodies were replaced with PBS to produce a negative control, and a known positive tissue slice was used as a positive control. Analysis of immunohistochemistry results Two pathologists who were blind to diagnosis independently inspected the slices. The rate of agreement between the two pathologists was 95%. The scores from both pathologists were averaged to provide Clostridium perfringens alpha toxin the final score for each case. A combination of positive cell count and staining intensity was used for scoring. Positive cell count was scored based on the average percentage of positive cells per 100 cells in 10 high-power fields, as follows: 0–10%, score 0; 11–25%, score 1; 26–50%, score 2; 51–75%, score 3; and >75%, score 4. Staining intensity was scored as follows: negative, score 0; faint yellow, score 1; yellow or deep yellow, score 2; brown or dark brown, score 3. The final score was obtained by multiplying the cell count and staining intensity scores. For VEGFR-2 and c-Met, a score of ≥ 5 was defined as high expression and a score of < 5 was defined low expression.