Observer 1 microscope

The harvested human or rat urethral tissues were fixed in 4% neutral phosphate-buffered paraformaldehyde overnight and processed using routine methods before sectioning into slices. The sections were stained with Hematoxylin-eosin (HE), Masson and Sirius stain, or were subjected to immunohistochemistry analysis. Representative images were captured using a Zeiss Observer I microscope (Zeiss) at magnifications of 20×, 50×, and 100× and acquired using the CaseViewer software.

Human or rat urethral tissues sections were deparaffinized and incubated in peroxidase solution (3% H₂O₂) followed by blocking at room temperature for 30 min with 3 % BSA. Next, slides were incubated with anti-β-catenin antibody (1:200, Abcam), anti-DKK1 antibody (1:500, Abcam) or anti-α-SMA antibody (1:500, Abcam) at 4°C overnight. Slides were then incubated with a second antibody conjugated to horseradish peroxidase (HRP) for 1 h and followed with diaminobenzidine (DAB) staining and neutral resin sealing. The representative images were captured using a Zeiss Observer I microscope (Zeiss) at magnifications of 20×, 50×, and 100× and collected using the CaseViewer software.