Sections were rinsed twice in ��1 PBS, acetylated for 10 min with 0.25% acetic anhydride/0.1 m triethanolamine, dehydrated in a graded series of ethanol/dietyl pyrocarbonate (DEPC)-treated water selleck products rinses and air dried. Hybridization was carried out overnight at 55��C with sense or antisense digoxigenin-labelled (labelling-kit; Roche Diagnostics, Basel, Switzerland) RNA probes (2 ��g/ml) diluted in hybridization buffer, which contained 50% deionized formamide, ��4 saline sodium citrate (SSC) (��1 SSC; 0.15 m NaCl, 15 mm sodium citrate, pH 7.0), 10% dextran sulfate, ��1 Denhardt’s solution, 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA and 10 mm dithiothreitol. Following hybridization, sections were washed in ��0.2 SSC for 50 min at 72��C and incubated with RNAse (20 ��g/ml) for 30 min at 37��C.
Hybridization signal was visualized using anti-digoxigenin-alkaline phosphatase-conjugated antibodies (Roche Diagnostics, Basel, Switzerland) and nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) as a substrate. Results AhR-null mice had important hepatic alterations. As it can be observed in Fig. 1, whereas hepatic portal fibrosis were markedly visible in AhR?/? mice, AhR+/+ mice showed no apparent liver damage (Fig. 1a,b). We have used collagen deposition around the periportal areas as a fibrotic marker in liver. Fig. 1(a,b) shows atrophy of the tissue surrounding the portal triad (arrow). Sirius red staining of AhR?/? liver sections reflected an increase in collagen content in the portal triads, which was not observed in AhR+/+ mice.
No significant collagen staining was found in other areas of the liver. The altered structure of the portal triads in AhR?/? mice also affected the diameter of the bile duct. Thus, whereas in AhR+/+ mice, the bile duct had a diameter of 54 �� 27 ��m (n = 7), in AhR?/? mice, this value increased to 106 �� 34 ��m (n = 7). Figure 1 Histology of normal and fibrotic mice livers. Serial 4-��m frozen sections containing a typical portal triad were prepared from wild-type AhR (AhR+/+) and null AhR (AhR?/?). (Panels A, B) Collagen was localized (red) by Fast green/Sirius … Because the fibrotic nodule was big enough, we could obtain several sections from the same area of the liver. Thus, the same fibrotic area was used to analyse ��-actin and vimentin protein expression, which should be selectively expressed by the fibroblasts present at the damaged site.
Immunostaining signals for ��-actin and vimentin Entinostat were markedly increased in AhR?/? with respect to AhR+/+ liver (Fig. 1c�Cf, respectively). Moreover, the expression of both proteins was limited to the fibrotic areas previously found to be positive for Sirius red staining (compare Fig. 1b,d,f). In addition, another relevant marker for fibrogenesis, fibronectin, was also increased in the portal region of AhR?/? liver (Fig. 1g,h).