Eclipse 80i light microscopy

Animals' tissues were fixed in 4% formaldehyde, dehydrated with gradient ethanol and embedded in paraffin. The tissue sections (4 µm) were dewaxed and rehydrated per a standard protocol. For histological analysis, the sections were stained with hematoxylin and eosin. Once cell got damaged, cells became edematous and deformed and finally got necrosis with its structure disorganized and nuclear fragmentation. And at least 3 tissue samples and 5 fields of view were analyzed to determine the cell damages in each experimental group. H&E assay could partly indicate cell damage but the damaged cells need a specific staining. So we performed TUNEL assay which can specifically distinguish apoptotic cells. For the TUNEL assay, an in situ apoptosis detection kit (Wuhan Boster Biological Technology, Ltd.) was used to detect apoptotic cells in heart tissues. The positive cells were identified, counted (three random fields per slide) and analyzed through ECLIPSE 80i light microscopy (Nikon, Tokyo, Japan).