Measuring Liver Fibrosis and VEGF-A

The expression of Sct-dependent microRNA 125b (that regulates IBDM and liver fibrosis through changes in the expression of biliary VEGF-A) was measured by qPCR^{9 (link)}. By immunohistochemistry, we evaluated the semiquantitative immunoreactivity for VEGF-A in liver sections (4–5 µm thick, 10 different fields analyzed from 3 different samples). When 0–5% of bile ducts were positive for VEGF-A, a negative score was assigned; a +/− score was assigned when 6–10% of bile ducts were positive; a + score was assigned when 11–30% of bile ducts were positive; a ++ score was assigned with 31–60% of bile ducts positive; and a score +++ was assigned when more than 61% of bile ducts were positive. The expression of: (i) VEGF-A/R-2 in cholangiocytes and total liver; (ii) IL-6 and TNF-α in cholangiocytes; and (iii) CD31 and vWF in total liver was evaluated by immunoblots and/or qPCR. Immunofluorescence for the expression of CD31 was performed in frozen liver sections (10 µm thick). Immunofluorescent staining was visualized using Leica TCS SP5 X system (Leica Microsystems Inc.).