Hydrochloric acid alcohol

The left ventricular anterior wall tissue of each group of rats was collected and immersed in 4% paraformaldehyde fixative for 24 h. The tissue was then dehydrated in gradient alcohol and placed in paraffin to make a paraffin mass. Then, we used a microtome (RM2235, Leica, Wetzlar, Germany) to make paraffin sections. We dewaxed and hydrated paraffin sections in xylene and gradient alcohol. The sections were then rinsed in running water for 3 min. The sections were placed in hematoxylin stain (Beyotime, Shanghai, China) for 1 min. After rinsing under running water, we placed the sections in hydrochloric acid alcohol (Beyotime, Shanghai, China) for another 3 s. The sections were then rinsed with running water, placed in eosin staining solution (Beyotime, Shanghai, China) for 1 min, and dehydrated to seal the sections.

Rat myocardial tissue was fixed with 4% paraformaldehyde at 4˚C. After 24 h, myocardial tissue was dehydrated and embedded in paraffin. A microtome was subsequently used to section the myocardial tissue (thickness, 5 µm). Sections were dewaxed, hydrated and stained with hematoxylin solution (Beyotime Institute of Biotechnology) for 1 min at 37˚C. After rinsing sections for 3 min with running water, excess stain was removed with hydrochloric acid alcohol (Beyotime Institute of Biotechnology). Samples were then immersed in eosin solution (Beyotime Institute of Biotechnology) for 1 min and dehydrated at 37˚C. Neutral gum was used to seal sections. Images were acquired using a light microscope (Nikon/80i; Nikon Corporation) and a digital camera (DP71CCD; Olympus Corporation).