Light green

Surgical tissue of human ileum and colon, fixed in Carnoy's fixative or HistoChoice™ Tissue Fixative (Sigma Aldrich, St. Louis, MO), were paraffin embedded and sectioned (4–5 μM) and mounted on X-tra™ positive-charged slides (Leica Biosystems, Buffalo Grove, IL). Sections were deparaffinized, rehydrated in gradient ethanol, H₂O, and PBS and blocked with 5% donkey serum (in 1 × PBS) for 30 min prior to overnight incubation at 4 °C with antibody^{51 (link)}. Following incubation, slides were washed, and probed with Vectastain ABC kit goat anti-rabbit/goat secondary antibodies (Vector Laboratories, Burlingame, CA), activated with 3,3-diaminobenzidine peroxidase (Vector Laboratories, Burlingame, CA), and counterstained in 0.4% light green (Sigma Aldrich, St. Louis, MO), 0.2% glacial acetic acid for 5 min prior to dehydration and mounting^{51 (link)}. Samples were visualized using an Olympus BX51 microscope (Center Valley, PA) and images were processed using Adobe Creative Suite software (version 25, Adobe Inc., San Jose, CA).

ASCs differentiation potential into adipocytes, osteoblasts, and chondrocytes, were evaluated in third passage with a commercial medium (Lonza, Walkersville, EUA). The medium inducer was changed every 3 days during 3 weeks. To adipogenic and osteogenic differentiation, cells were seeded on glass coverslips (Sarstedt, Newton, NC, USA) in 24-well plates (Sarsted). Briefly, cells were treated with Bouin's fixative (Biotec, Labmaster, Paraná, Brazil) for 10 min, washed twice with 70% ethanol and once with Milliq water. Oil Red O (Sigma-Aldrich) was used to visualize lipid-rich vacuoles and hematoxylin-eosin (HE) (Biotec) was used for nuclear staining. Osteogenic differentiation was evaluated by Alizarin Red S (Fluka Chemie, Buchs, UK) and light green (Sigma-Aldrich) was used to counterstain. For chondrogenic differentiation, cells were grown in micromass culture. Briefly, 5 × 105 cells in 0.5 ml of medium were centrifuged at 300 g for 10 min in a 15 mL polypropylene tube to form a pellet. Without disturbing the pellet, cells were cultured for 21 days with medium inducer. On day 21, cell aggregates were fixed in 10% formaldehyde for 1 h dehydrated in serial ethanol dilutions and embedded in paraffin blocks. Toluidine Blue staining (Sigma-Aldrich) demonstrate the presence of intracellular matrix proteoglycans. Control cells were kept in DMEM-F12 medium with 15% FCS.

Paraffin-embedded sections were deparaffinized and rehydrated as described above. Sections were then analyzed for in situ cell death detection using the In Situ Cell Death Detection Kit, AP (Roche Applied Science, Germany). Following manufacturer’s instructions, positive control slides were created by applying recombinant DNase I for 10 minutes at room temperature to induce DNA strand breaks prior to the TUNEL labeling procedure. Negative control slides were incubated with 50 μL of label solution lacking terminal transferase. Positive control and treatment group slides were incubated with 50 μL of TUNEL reaction mixture containing Converter-AP and substrate solutions. Following TUNEL staining, slides were counterstained with 0.5% Light Green (Sigma).

The slides were placed back to back in Coplin jars and fixed with freshly prepared cold methanol and a glacial acetic acid mix (3:1). The slides were air dried at room temperature for 10 min, then stained with 5 M 37% (v/v) in hydrochloric acid at room temperature for 30 min, according to the established Feulgen technique [30 (link)]. The slides were rinsed with running tap water for 3 mins, then stained with Schiff’s reagent (Sigma-Aldrich, Steinheim, Germany) at room temperature and under dark conditions for an additional 90 min. The slides were washed for 5 min with running tap water, counterstained with 0.2% (w/v) Light Green (Sigma-Aldrich, Steinheim, Germany) cytoplasmic stain for 20 s, and were then washed again with running tap water.