Phosphate buffered saline (pbs)

After harvesting, organs were fixed in 4% paraformaldehyde (PFA) solution in PBS (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) for 1 day at 4 °C, immersed in a 30% (w/v) sucrose solution in PBS for 2 days at 4 °C, and frozen by vapor of liquid nitrogen, then stored at −80 °C until sectioning. Organs were embedded into Frozen Section Compound (Leica FSC 22 Clear, Leica Microsystems srl, Italy) and 20 µm slices were prepared using cryostat instrument. The sections were then placed on HistoBond® microscope slides (Marienfeld, Germany) and stained with Haematoxylin‐Eosin (Modified Gill's Type II Haematoxylin; Eosin G) or 4’,6‐diamidion‐2‐phenylindole (DAPI) and Luciferase antibody (Primary Antibody, Red Firefly Luciferase Polyclonal Antibody, eBioscience, 1:50; Secondary Antibody, Goat Anti‐Rabbit IgG Cross‐Adsorbed Alexa 594, Invitrogen, 1:1000) for tumor recognition. Slices were imaged using confocal microscopy (Nikon A1).

C2C12 cells were fixed in 4% paraformaldehyde in PBS (Santa Cruz, Dallas, TX, USA) for 15 min and permeabilized with 0.5% Triton-X in PBS for 15 min at room temperature. To avoid non-specific binding, cells were blocked with 5% BSA in PBS for 30 min. Immunolabeling with anti-Myosin Heavy Chain (MHC) primary antibody (clone MF20, Developmental Studies Hybridoma Bank) in 5% BSA was done overnight at 4°C. Secondary anti-mouse antibody coupled to Alexa Fluor 555 (Molecular Probes) was used to detect MHC signal in myotubes. Nuclei were counterstained with Hoechst 33258 (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA). Images were taken with Olympus fluorescent microscope with 20x magnification.

To perform the histochemical assessment, scaffolds were fixed in 4% formaldehyde solution in PBS (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min and, washed in PBS 1× twice. Samples were then dehydrated by a graded series of ethyl alcohols (from 50% to 100%), clarified by xylene for 45 min twice and incubated in paraffin overnight at 56 °C to allow paraffin infiltration. After two incubations in paraffin, samples were embedded in paraffin blocks horizontally. Ten-micrometre-thick slices were cut and, after deparaffination and rehydration, Haematoxylin and Eosin and Sirius Red staining were performed. Haematoxylin and Eosin staining was observed by light microscope, while Sirius Red staining by both light and fluorescence microscope. The homogeneity PLGA_TGF-β1 NPs distribution throughout the composite scaffold was investigated by immunohistochemistry on 10-µm-thick paraffin-embedded sections collected at different Z-axis depths. Deparaffination and rehydration with xylene and a graded series of ethyl alcohols (from 100% to 50%), antigen retrieval, primary antibody incubation, antigen visualization, and section mounting were performed as described above. Four images at 10× magnification were captured from each slice and examined for semi-quantitative analysis as disclosed above.