Sirius red

Kidney tissues were cut into 4-μm paraffin sections and stained with Sirius red (Abcam, Cambridge, UK) to observe their histology. Deparaffinization and hydration were performed using xylene and ethanol, respectively. To block endogenous streptavidin activity, 3% hydrogen peroxide (H₂O₂, Sigma-Aldrich, St. Louis, MO, USA) was used. The deparaffinized sections were stained with anti-mouse TG2 antibody (Novus Biologicals, Centennial, CO, USA). These sections were then incubated with secondary antibodies, goat anti-rabbit (Vector Laboratories, Burlingame, CA, USA) and rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA, USA). The sections were then counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and visualized under a light microscope (DFC-295; Leica, Mannheim, Germany). For rodent and human kidney samples, at least five fields (magnification: ×100) were randomly selected, and Sirius red and brown-stained areas were quantified using computer-based morphometric analysis (Qwin 3; Leica).

All histological procedures were performed using standard procedures at the histology core facility of the Goodman Cancer Research Centre (McGill University). Liver tissues were fixed in 4% formaldehyde for 2 days at room temperature immediately after sacrifice, embedded in paraffin, and cut to 4-μm sections on slides using a Leica Microtome (Leica Biosystems). The slides were stained with hematoxylin and eosin (Leica Biosystems, cat# 3802098), or Sirius Red (Abcam, cat# ab150681) using a Leica ST5020 Multistainer (Leica Biosystems) according to the standard protocol. Stained images were acquired using an Aperio ScanScope XT digital scanner (Leica Biosystems) and analyzed with ImageScope software (version 12.3.3.5048, https://www.leicabiosystems.com/fr/imagerie-pathologique/integrer/aperio-imagescope/).

After sacrificing the mice on day 28, left lung tissues were fixed in 10% formalin, embedded in paraffin and cut into 4 μm thick sections. Sections were stained with hematoxylin and eosin (Sigma-Aldrich Inc., St. Louis, MO, USA) or Sirius Red (Abcam, Cambridge, UK).

Histology of the muscle was performed by Electron Microscopy and Histology Core of Augusta University. After 7 days or 30 days of cell transplantation, the TA muscle was harvested and embedded in paraffin. Five-micrometer-thick sections of the TA muscle were cut and stained with hematoxylin and eosin (H and E), Masson's trichrome, and Sirius red according to the manufacturer’s protocol (Abcam). Images were taken by a vertical microscope (Olympus, Japan). Fibrosis and necrosis were determined using the ImageJ software (NIH) and expressed as the ratio of the total area of the cross-section and normalized with the ratio of the control lateral TA muscle section. Myofiber necrosis was identified with fragmented sarcoplasm [15 (link)] and/or increased inflammatory cell infiltration and was measured using non-overlapping tile images of transverse muscle sections.