α sma

Paraffin-embedded sections were rehydrated and antigen retrieval for Ki67 was performed by microwaving in 10 mmol/L sodium citrate (pH 6.0) for 10 minutes. Frozen sections were fixed with acetone and stained for alpha smooth muscle actin (α-SMA) (Sigma-Aldrich, St Louis, MO). α-SMA primary antibodies were incubated at 4 °C overnight, whereas Ki67 (Lab Vision, Freemont, CA) primary antibody was incubated for 1 hour at room temperature. Signal was enhanced using the tyramide amplification system (PerkinElmer, Boston, MA).

Tissue sections were deparaffinized and subsequent blockage of the endogenous peroxidase activity was achieved by incubation with 2.5% methanolic hydrogen peroxide for 30 min. The tissue sections were stained with rabbit antibodies against Ki-67, MMP13, collagen I, desmin (GeneTex, Irvine,CA) or TIMP-1 (Proteintech Group Inc, Chicago, USA) by using the Mouse/Rabbit Probe HRP Labeling Kit (BioTnA Biotech, Kaohsiung, Taiwan). Binding of mouse monoclonal antibodies against GNMT (14-1, YMAC Bio Tech, Taiwan), α-SMA (Lab Vision, Fremont, CA) and TGF-β1 (R&D Systems) was detected by using the N-Histofine Mousestain Kit (Nichirei Biosciences, Tokyo, Japan). The immunohistochemical assays were performed according to the manufacturer’s instructions. The sections were developed with 3-3′ diaminobenzidine (DAB) (Dako, Milan, Italy) and finally counterstained with hematoxylin (Dako). Negative controls were performed using normal mouse antiserum instead of the primary antibody.