1989; Peterson selleck Bortezomib 1993). Briefly, liver sections were incubated in 0.04% solution of Fast green (Sigma-Aldrich Chemical, St Louis, MO, USA), in saturated picric acid for 15 min at room temperature. The sections were then washed with distilled water, incubated for 30 min in Fast green 0.04% and Sirius red 0.1% (Sigma-Aldrich Chemical), also in saturated picric acid, and washed again with distilled water. Immunohistochemistry Liver cryo-sections were permeabilized with 0.05% Triton X-100 diluted in phosphate-buffered saline (PBS-T). Endogenous hepatic peroxidase activity was blocked by treatment with H202 for 45 min at room temperature (1% H202 diluted in PBS-T). Tissues were then rinsed twice in a solution of 0.2% gelatin and 0.25% Triton X-100 in PBS (PBS-GT) for 15 min.

Nonspecific binding sites were blocked by 1-h incubation in 0.1 m lysine, 0.2% gelatin and 0.25% Triton X-100 in PBS. Sections were incubated overnight at room temperature with primary antibodies against smooth-muscle ��-actin (Master Diagnostica Clone 1A4, Badalona, Barcelona), vimentin (Developmental Studies Hybridoma Bank, H5, Universitty of Iowa, IO, USA), fibronectin (Developmental Studies Hybridoma Bank, B3/D6), LTBP-1 (Dallas et al. 2000) and TGF-��1 (R&D, clone 1D11, Minneapolis, MN, USA) diluted at 1 : 100 in PBS-GT. After complete washing with PBS-T and PBS-GT, sections were incubated for 2 h with secondary antibodies conjugated to peroxidase (antigoat for ��-actin, antimouse for vimentin, fibronectin and TGF-��, and antirabbit for LTBP-1) diluted at 1 : 200 in PBS-GT.

Colour development was induced using diaminobenzidine (DAB) as a substrate (0.025% DAB w/v, 0.06% H202 v/v in PBS). LTBP-1 and TFG-��1 riboprobe cloning To synthesize the riboprobes, cDNA fragments of LTBP-1 and TGF-��1 were first generated by reverse transcriptase-PCR (RT-PCR) and cloned. One microgram of total RNA obtained from wild-type mice was reverse transcribed by using oligo-dT priming. Aproximately, 3 ��l from the RT reaction were amplified in 25 ��l of reaction mixture containing 1.5 mm MgCl2, 0.2 mm dNTPs, 2.5 units of Taq polymerase (Ecogen SRL, Barcelona, Barcelona) and 25 pmol of each primer forward (EcoRI site underlined) 5��-CGGAATTCCGGGAGTGCTATTATAACCTCAATG-3�� and reverse (BamHI site underlined) 5��-CGGGATCCCGGGGTCTTGGCATTCATCCAT-3��.

Cycling conditions were 94 ��C for 2 min, and then 35 cycles of 94��C for 1 min, 58��C for 1 min and 72��C for 2 min, followed by a 5 min final extension at 72��C. For TGF-��1, conditions were identical except for the primers used; forward (EcoRI site underlined): 5��-CGGAATTCCGATC-CTGTCCAAACTAAGGCTC-3�� GSK-3 and reverse (BamHI site underlined): 5��-CGGGATCCCGCGTCAAAAGACAGCCAC-TCAG-3��. RT-PCR fragments were ligated into pGEM vectors containing SP6 and T7 promoters. In situ hybridization In situ hybridization was carried out essentially as described (Corchero et al. 1999).