Envision hrp antimouse

Urinary bladders were fixed in 10% neutral-buffered formalin, dehydrated through a graded ethanol series, and embedded in paraffin. Embedded tissues were sectioned at a thickness of 8 µm. The tissue samples were processed routinely for standard staining with hematoxylin and eosin (H&E). To confirm the smooth muscle regeneration, an immunohistochemical staining with anti-smooth muscle α-myosin heavy chain (α-SMM) antibody was used. Briefly, the tissue sections were incubated with primary antibody against α-SMM (dilution 1:400; Abcam, Great Britain). After washing, the sections were overlaid with peroxidase-conjugated antimouse secondary antibody (EnVision⁺/HRP antimouse; Dako). Twelve tissue sections for each experimental group (3 urinary bladders × 4 tissue section each) were examined. Stained samples were analyzed using light microscopy by 2 independent pathologists. Additionally, the samples were evaluated for smooth muscle content using the ImageJ software according to the method described previously.⁷ Analysis was repeated for 5 areas from each specimen.

To verify multipotency of ADSCs and BM-MSCs, the cells were differentiated in vitro into adipogenic, osteogenic, and chondrogenic lineages. The differentiation was induced by culture in appropriate differentiation media, according to the manufacturer’s instructions (Invitrogen, USA). Adipogenesis was measured by the accumulation of neutral lipids in fat vacuoles and stained with Oil Red O (Sigma-Aldrich). Osteogenesis was confirmed using Alizarin Red staining (Millipore, USA). Chondrogenic differentiation was evaluated by anticollagen type II immunocytochemical staining (anticollagen type II clone 6B3, dilution 1:100; Millipore and EnVision⁺/HRP antimouse; Dako, Denmark). Stained samples were analyzed using light microscopy by 2 independent pathologists.