Protective Effect and Mechanism of Alprostadil in Acute Respiratory Distress Syndrome Induced by Oleic Acid in Rats

Yan, Xiujuan; Li, Yingxiu; Choi, Yun Ho; Wang, Chongyang; Piao, Yihua; Ye, Jing; Jiang, Jingzhi; Li, Liangchang; Xu, Huixian; Cui, Qingsong; Yan, Guanghai; Jin, Minggen

Lung sections of 5 μm were kept in an oven at 60°C for 24 h to increase section adherence to the slide. After deparaffinization and rehydration, the sections were treated with 10 mM citrate buffer (Cat #ZLI-9064, ZSGB-BIO, Beijing, China) for 5 min for antigen retrieval. Then, the sections were incubated with 3% H 2 O 2 for 10 min to eliminate endogenous peroxidase activity, followed washing 3 times with PBS or TBS for 2 min each time. Sections were then washed with PBS 3 times for 2 min each time. We then added rabbit anti-rat anti-ACE antibody (1: 300 dilution, Elabscience Biotechnology Co., Ltd, China), followed by incubation at 4°C overnight. Sections were then washed 3 times with PBS or TBS for 2 min each time. We added reagent 1, then incubated sections at room temperature for 20 min, followed by washing 2 times with PBS for 2 min each time. Reagent 2 was then added, followed by incubation at room temperature for 20~30 min and washing 3 times with PBS for 2 min each time. Color development was performed with DAB (Cat # ZLI-9019, ZSGB-BIO, Beijing, China). We washed sections with deionized water to make the section counterstained, dewatered, and transparent, then sealed the section and viewed it under the microscope. Under 200× magnification, 5 different microscopic fields were randomly chosen. Quantitative analysis of ACE expression was performed by calculating the average optical density of ACE-positive staining.