Masson stain

The HA/TCP grafts loaded with MSCs and bone tissues were collected, fixed in 4% PFA, and then decalcified for 10 days in 10% EDTA (pH 7.4). After decalcification, the specimens were dehydrated and subsequently embedded in paraffin. Sections (5 mm thickness) were stained with HE stain (catalog no. AR1180; Boster) and Masson stain (catalog no. G1340-100; Solarbio) according to the manufacturers’ instructions. The slides were subjected to immunohistochemistry with anti-collagen I (catalog no. ab34710; Abcam).

Heart tissues were fixed with 4% paraformaldehyde at 25°C for 72 h, paraffin-embedded, and cut into 5-μm-thick sections. Heart sections were stained with hematoxylin and eosin, Masson stain, and Sirius Red stain (all from Solarbio, Beijing, China). Paraffin-embedded sections were dewaxed, blocked, and incubated with rabbit anti-4 hydroxynonenal antibodies (4HNE, 1:500; R&D, Shanghai, China) overnight at 4°C under humidified conditions. Subsequently, sections were incubated with HRP-labeled secondary antibodies (1:2000) at 25°C for 1 h. Diaminobenzidine (DAB) was used for visualization, and images were captured under an inverted Olympus IX70 microscope (Olympus, Beijing, China). For histological analysis, the cross-sectional areas of myocytes and tissue sections were stained with wheat germ agglutinin (WGA; plasma membrane staining, Alexa Fluor 488 conjugated) and analyzed using the ImageJ software (NIH, Maryland, USA).