Frozen gastrocnemius muscles were sectioned at 10 μm thickness using a cryostat and subjected to immunohistochemistry as described above [14 (link)].
For Ki67 staining, cells cultured under indicated condition and treated with salidroside or PBS were seeded in a 15-mm glass bottom cell culture dish, and then further cultured for 12 h. Cells were fixed with 4% paraformaldehyde and permeabilized for 5 min with PBS containing 0.1% Triton X-100. After blocking with 1% bovine serum albumin for 1 h, the samples were incubated at room temperature for 90 min with an anti-Ki67 antibody and subsequently, stained with secondary antibodies anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies). DAPI (Beyotime, Guangzhou, China) was used to stain nuclei. Images were taken with Microsystems-AF6000 (Leica, Heidelberg, Germany). For phalloidin staining, samples were incubated at room temperature for 30 min with phalloidin after blocking, and images were taken with Microsystems-TCS SP5 (Leica). The quantification of F-Actin formed from G-Actin polymerization was performed by fractal dimension as described previously [25 (link)], by using ImageJ software. For experiments with conditioned media, the cells were cultured with conditioned medium under hypoxia for 12 h prior to staining. The antibodies used were listed inSupplementary Table 2 .
For Ki67 staining, cells cultured under indicated condition and treated with salidroside or PBS were seeded in a 15-mm glass bottom cell culture dish, and then further cultured for 12 h. Cells were fixed with 4% paraformaldehyde and permeabilized for 5 min with PBS containing 0.1% Triton X-100. After blocking with 1% bovine serum albumin for 1 h, the samples were incubated at room temperature for 90 min with an anti-Ki67 antibody and subsequently, stained with secondary antibodies anti-rabbit Alexa Fluor 488 (Invitrogen Life Technologies). DAPI (Beyotime, Guangzhou, China) was used to stain nuclei. Images were taken with Microsystems-AF6000 (Leica, Heidelberg, Germany). For phalloidin staining, samples were incubated at room temperature for 30 min with phalloidin after blocking, and images were taken with Microsystems-TCS SP5 (Leica). The quantification of F-Actin formed from G-Actin polymerization was performed by fractal dimension as described previously [25 (link)], by using ImageJ software. For experiments with conditioned media, the cells were cultured with conditioned medium under hypoxia for 12 h prior to staining. The antibodies used were listed in
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