Immunofluorescent staining was performed

to reveal the colocalization of integrin MAPK Inhibitor Library cell line αvβ3 with α-SMA (aHSCs), albumin (HC), CD31 (vascular endothelial cells), CD68 (macrophages), and CD163 (Kupffer cells) in the liver sections. There is no specific antibody against rat integrin αvβ3 available. To date, the majority of β3 has been shown to bind to αv (αvβ3) or αIIb (αIIbβ3), and the latter is a membrane receptor expressed only in cells of megakaryocytic lineage and some tumor cells.12, 24 Hence, evaluating positive immunofluorescent staining of the β3 subunit represents the positivity of integrin αvβ3. Primary antibodies against polyclonal anti-β3 integrin (1:200; Chemicon, Billerica, MA), monoclonal anti-SMA (1:400; Chemicon), RO4929097 in vitro polyclonal anti-albumin (1:50; AbD Serotec, Oxford, UK), monoclonal anti-CD31 (1:50; AbD Serotec), monoclonal anti-CD68 (1:50; AbD Serotec), and monoclonal anti-CD163 (1:50; AbD Serotec) were used. Secondary antibodies included fluorescein isothiocyanate (FITC)-conjugated IgG (1:200) and Cy3-conjugated IgG (1:200). 0.2% Triton X-100 was used for permeabilization when appropriate. DAPI was used for nuclear counterstaining.

Multicolored fluorescent staining of liver sections was analyzed by confocal laser scanning microscopy (Leica Microsystems, Wetzlar, Germany). The fluorescent signals of liver sections were video-digitized and analyzed with a software program that automatically outlined the total stained areas with threshold setting (Photoshop 4.0; Adobe).25 These areas were then quantified with NIH Image 1.62 software and the percentage of the merged yellow color region to the total integrin αvβ3-stained green region in each section was calculated. Ten randomly selected amplifying Amino acid fields (400×) in each section were assessed.

The hepatic messenger RNA (mRNA) levels of αv, β3 integrin subunits and α-SMA were quantitated using qRT-PCR analysis as described.26 All PCR primers (Table 1) were designed by Primer Premier 5.0 using published rat gene sequences obtained from the National Center for Biotechnology Information database. The hepatic protein amount of rat αv, β3 integrin subunits and α-SMA was determined by western blot analysis as described.18 The liver sections of TAA-treated or control rats (n = 8 per group) were used to visualize 125I-cRGD binding to livers as described.27 In brief, the liver sections were incubated in Tris-HCl buffer containing 100 pmol/L 125I-cRGD at 4°C for 24 hours. At the same time, the parallel sections were incubated in the buffer mixed with 100 pmol/L 125I-cRGD and 5 μmol/L cRGD to verify whether the excess cRGD would block the binding of 125I-cRGD in liver sections. After incubation, radioautographic films (Amersham, Buckinghamshire, UK) were exposed to labeled sections. After exposure and developing, the films were scanned with an automatic imaging analyzer and the relative absorbance of hepatic historadioautography was measured.