Tanshinone IIA Attenuates Bleomycin-Induced Pulmonary Fibrosis via Modulating Angiotensin-Converting Enzyme 2/ Angiotensin-(1-7) Axis in Rats

Wu, Huajie; Li, Yan; Wang, Yanxia; Xu, Dunquan; Li, Congcong; Liu, Manling; Sun, Xin; Li, Zhichao

Lung sections of 4-μm were prepared and deparaffinized, rehydrated, and blocked by incubating in 0.3% H 2 O 2 for 30 min. After antigen retrieval was performed the slides were incubated for 1 h with normal goat serum, and then incubated with TGF-β1 mouse monoclonal antibody, ACE-2 mouse monoclonal antibody, and ANG-(1-7) mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4 ℃ overnight. After washed in phosphate buffered saline (PBS), the tissues were incubated with corresponding antibody for 30 min at 37 ℃. Diaminobenzidine (DAB) signal detection method was performed for 8 min at room temperature. Negative controls were performed using mouse serum as the primary antibody. Positive staining was colored in deep brown. All sections were evaluated using an Olympus BX50 optical microscope equipped with an image analysis program (Image Pro Plus version 6.0, Immagini e Computer, Milan, Italy) and analyzed quantitatively. The integrated optical density (IOD) was calculated for arbitrary areas, measuring in 10 fields for each sample using a 40× objective.