2.3 | Immunohistochemistry
The LF specimens were fixed in 10% neutral formalin, embedded in paraffin and finally cut into sections with the thickness of 4μm. After dewaxing in xylene and rehydrating in a series of alcohol solutions, the sections were firstly incubated with primary antibodies against Gli1, Shh, collagen I, collagen III and α-SMA (Abcam) at an optimum dilution recommended by the manufacturers overnight at 4℃. Subsequently, these sections were incubated with the respective secondary antibody (Abcam) at room temperature. Immunohistochemical analysis was performed and visualized by confocal laser scanning microscopy (ZEISS, German).