Ds u1

Serial 10 μm cross sections of the right muscle were obtained on a cryostat (CM510; Leica, Wetzlar, Germany) at −20°C. The sections were warmed to room temperature (RT) and then preincubated in 1% normal goat serum (EMD Millipore, Billerica, MA) in 0.1 M phosphate buffered saline (PBS; pH 7.6) at RT for 10 min. The primary monoclonal antibody was then applied: either (1) fast myosin (1 : 2000; Sigma, St. Louis, MO), which specifically reacts with the myosin heavy chain- (MHC-) IIa and IIx, or (2) SC-71 (1 : 1000; Developmental Studies Hybridoma Bank, Iowa City, IA), which specifically reacts with MHC-IIa. The sections were incubated in these primary antibodies overnight at RT and incubated with a secondary antibody (goat anti-mouse IgG) conjugated with horseradish peroxidase (HRP, Bio-Rad, Hercules, CA, 1 : 1,000) at RT for 3 hours. Diaminobenzidine tetrahydrochloride was used as a chromogen to localize HRP. Images of the stained muscle fibers were recorded with a photomicroscopic (E600; Nikon, Tokyo, Japan) image processing system (DS-U1; Nikon). The fibers were classified as Type I, IIa, or IIx/b fibers based on their immunohistochemical staining properties, and population and cross-sectional areas (CSAs) of each muscle fiber type were calculated in deep and superficial portions (Figure 1(a)).

PFA‐fixed samples were embedded in paraffin and sectioned at 4 µm. Sections of before and after decellularization samples were stained with hematoxylin and eosin (H&E) or 4′,6‐diamidino‐2‐phenylindole (DAPI; Vectashield, Vectorlabs). H&E stained slides were imaged with Zeiss Axiokop 20 microscope and captured with a Nikon DS‐U1 camera. DAPI stained slides were analyzed using EVOS microscope (Thermo Fisher Scientific).
Immunohistochemistry (IHC) staining was performed on before and after decellularization samples with Collagen Type I and Collagen type IV (Table S4). Antigen retrieval was performed in citrate buffer (pH = 6.0) at subboiling temperatures for 10 min. Primary antibodies were incubated over night at 4°C. Envision + system horseradish peroxidase antirabbit secondary antibody (DAKO) was incubated at RT for 60 min, before staining with 3'‐diaminobenzidine and and counterstaining with hematoxylin.

Transfected cells were inoculated into 6-well plates for 36 h at 37 °C under 5% CO₂. After reaching confluence, a sterile 20-μL pipette tip was used to wound the cell monolayer. The cells were then washed with PBS and incubated in DMEM containing 1% FBS. A Nikon inverted microscope (ECLIPSE TE-2000U), equipped with a video camera (DS-U1, Nikon), was used to capture images 0 and 24 h after wounding.

Paraformaldehyde-fixed kidney was used to prepare paraffin-embedded tissue sample. The paraffin-embedded tissue sections (5-μm thick) were stained with hematoxylin-eosin (H&E) or immunochemistry staining (IHC) (anti-CD68 mAb, Cat. ab31630, Abcam, USA), and observed under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan). For histopathological assessment, representative images were captured with a camera (DS-U1, Nikon, Tokyo, Japan) and analyzed with the ACT-2U image analysis software (version: 1.40.85.221, Nikon, Tokyo, Japan). Tubular injury was evaluated independently by two pathologists blinded to the treatments. Ten fields were randomly selected at a magnification of 400× and 10 tubules were selected from within each field. The scoring was performed as previously described31 (link). Briefly, slides were evaluated based on the presence and extent of tubular epithelial cell flattening (1 point), brush border loss or shedding (1 or 2 points), the presence of a cast (2 points), and caducous necrotic cells without formation of cast or cell debris (1 point). For IHC assessment, ten fields were randomly selected from the slide of each animal at a magnification of 400×, and the number of positively stained cells was counted. Six rats were evaluated for IHC staining in each group.

For immunofluorescence staining of 5 µm placenta sections, the detailed procedure is shown in Duan et al., 2021 [36 (link)]. The antibodies used are listed in Supplementary Table S2. The stained sections were visualized under a confocal fluorescence microscope (Leica SP5, Wetzlar, Germany) and photographed with LAS AF software version 4.0 (Leica, Wetzlar, Germany). A minimum of three placental tissues for each condition was investigated.
For immunohistochemical (IHC) staining, the tissue sections were subjected to routine dewaxing, antigen retrieval, and blocking. Subsequently, tissue sections were incubated in 3% H₂O₂ in methanol for 10 min to quench endogenous peroxidase activity. Primary antibodies were left to incubate overnight at 4 °C followed by a 1 h incubation with secondary antibodies at 37 °C. Antibodies are shown in Supplementary Table S2. Biotin and horseradish peroxidase-conjugated streptavidin were incubated to amplify the target antibody signal. Subsequently, the sections were counterstained with hematoxylin, dehydrated with gradient ethanol, purified with xylene, mounted, and observed under a ZEISS Axiophot microscope. The sections were finally photographed by a digital camera (DS-U1, Nikon, Tokyo, Japan) with NIS-Elements BR 3.0 software.