Mounting medium

The heart sections were immersed in Maeda’s resorcinol fuchsin stain solution (Muto Pure Chemicals Co., Ltd.) for 1 h and washed with 100% ethanol. Heart sections (5-μm thick) were mounted on glass slides and immersed in Weigert’s iron hematoxylin solution and washed with Milli-Q water. The specimens were then immersed in Picro–Sirius Red solution (ScyTek Laboratories, Inc., West Logan, UT, USA) for 15 min, air-dried, and rinsed with xylene. After staining, the tissue sections were dehydrated in 70%, 95%, and 100% ethanol solutions and ethanol/xylene mixed solution, immersed in xylene four times, and sealed using a mounting medium (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). The sections were observed under a bright-field microscope BX53, (Olympus, Tokyo, Japan) using the cellSens software version 1.18 (Olympus) and BZ-X700 microscope (Keyence, Osaka, Japan). The collagen fiber content in the compact layer and cardiomyocyte density were measured using the Fiji software version 2.3.0 [75 (link)].

To observe the GAG content in the cartilage, the tissue slides were immersed in hematoxylin (Surgipath Medical Industries, Richmond, IL, USA) for 5 min, washed with 1% HCl (Merck, Darmstadt, Germany) and distilled water, immersed in 0.02% fast green (Merck, Darmstadt, Germany) for 1 min, washed with 1% acetate (Merck, Darmstadt, Germany) and distilled water, immersed in safranin O (Merck, Darmstadt, Germany) for 10 min, washed with 95% and 100% ethanol, and finally sealed with mounting medium (Muto Pure Chemicals, Tokyo, Japan).