Vectershield anti fading mounting medium

In brief, we used the One Step TUNEL Apoptosis Assay Kit (Beyotime, C1088) according to the instruction provided by the manufacturer. Paraffin-embedded sections were deparaffinized in xylene and rehydrated in gradient ethanol (100%, 100%, 95%, 80%, 75%). Proteinase K was diluted with 10 mmol/L Tris-HCl (1:1000), then sections were incubated at RT for 30 min and rinsed with PBS. Next, the sections were stained with TUNEL working solution at 37°C for 1 h, followed by incubation with Hoechst 33342 (Thermo Fisher, H3570, 1:1000) to visualize the nucleus. Finally, the sections were mounted in VECTERSHIELD^® anti-fading mounting medium (Vector Laboratories, h-1000), and images were obtained using Zeiss LSM900 confocal system. Image J was used to quantify the percentage of TUNEL-positive cells.

Immunofluorescence staining was performed as previously described.⁸⁵ Paraffin-embedded sections were deparaffinized in xylene and rehydrated through gradient alcohol (100%, 100%, 90%, 80%, 70%, 50%). After rinsing in distilled water, sections were microwaved in 10 mM sodium citrate buffer (pH 6.0) three times for 2 min each. Upon cooling down to RT, sections were rinsed three times in PBS, permeabilized with 0.4% Triton X-100 in PBS for 30 min and rinsed again in PBS three times. Sections were then incubated with blocking buffer (10% donkey serum in PBS) at RT for 1 h, followed by incubation with primary antibodies overnight at 4 °C and fluorescence-labeled secondary antibodies at RT for 1 h. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific) before the sections were mounted in VECTERSHIELD anti-fading mounting medium (Vector Laboratories, h-1000). Images were obtained using a confocal laser scanning microscope (Leica TCS SP5 II). Antibodies used for immunofluorescence analysis are listed in Supplementary information, Table S7.

Immunofluorescence staining was performed as previously described with slight modifications.²³ For tissues, the muscle tissue embedded in the O.C.T. were cut into 10 μm cryosections by the Leica CM3050S cryostat. The cryosections were air‐dried for 15 min and washed three times in PBS before fixed with 4% PFA for another 20 min. Next, these sections were permeabilized with 0.4% Triton X‐100 in PBS for 1 h, and again rinsed with PBS three times. Cells were fixed with 4% PFA for 20 min and rinsed with PBS twice, and permeabilized with 0.4% Triton X‐100 (Sigma‐Aldrich) for 1 h at room temperature (RT). After blocking for 1 h at RT, the sections or cells were incubated with primary antibodies overnight at 4°C. Subsequently, the samples were washed several times with PBS and incubated with fluorescently labelled secondary antibodies for 1 h at RT. The nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific), and washed three times in PBS and then mounted in VECTERSHIELD anti‐fading mounting medium (Vector Laboratories). The image was acquired using a confocal laser scanning microscope (Leica TCS SP5 II). The antibodies used for immunofluorescence analysis are listed in Table S3.