Masson staining

Heart tissues were fixed using 10% formaldehyde for 24 h at room temperature (pH=7.2; cat. no. G2161; Beijing Solarbio Science & Technology, Co., Ltd.), decalcified, dehydrated, permeabilized using xylene (50% xylene for 1 h and 100% xylene for 2 h), embedded in wax and then sliced into 5 µm thick sections using a microtome. All the following steps were carried out at room temperature. The tissues were routinely deparaffinized and rehydrated in accordance. Weigert's hematoxylin (5%; cat. no. G1340; Beijing Solarbio Science & Technology, Co., Ltd.) was used to dye the cell nucleus for 5 min. Following rinsing with distilled water three times, the sections were stained using 0.7% Masson-Ponceau-acid fuchsin solution (cat. no. G1340; Beijing Solarbio Science & Technology, Co., Ltd.) for 10 min. Samples were then rinsed in 2% glacial acetic acid and differentiated in phosphomolybdic acid for 4 min. The sections were directly stained with 2% aniline blue dye solution (cat. no. G1340; Beijing Solarbio Science & Technology, Co., Ltd.). Following dehydrating with ethanol series, clearing with xylene and mounting with neutral resins, digital images were captured using a light microscope (magnification, ×200; Leica DM 4000B; Leica Microsystems, Inc.) to analyze fiber formation in the early callus.