2.8. Effect of Diannexin on Apoptosis in ARDS Rats

Apoptosis of ARDS-induced lung tissue injury was assessed using the TUNEL assay (Roche Diagnostics GmbH, Science, Mannheim, Germany). Briefly, each 4 μm lung tissue section was immersed in proteinase K and then washed with phosphate-buffered saline (PBS). Next, the sections were first immersed in blocking buffer at room temperature for 10 min and then immersed in TUNEL reaction solution for 1 h at 37°C. After washing with PBS, endogenous peroxidase activity was inhibited by H2O2, and then, the sections were incubated in the extra-avidin peroxidase and diaminobenzidine solution. Subsequently, the lung tissue sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted. An independent pathologist blinded to this study counted the number of apoptotic cells (brown nuclei) and total cells. The apoptosis index was calculated as the ratio of the number of apoptotic cells to total cells.

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