Immunofluorescence Staining of Nerve Fibers

The bone fixation method was consistent with HE staining. The bone slice thickness of 8.0 μm was rinsed with 0.05% PBST and Proteinase K (BOSTER, WuhFan, China) incubation antigen-repaired for 15 min at room temperature. The following experimental method of immunofluorescence staining was referred to our previous protocol (42 (link)). Briefly, the sections were incubated with the primary antibodies rabbit anti-Th (1:50, BOSTER, Wuhan, China) overnight at 4°C. After a 0.05% PBST rinsed, the sections were incubated with Alexa Fluor 594-labeled anti-rabbit IgG (1:400, Abcam, Cambridge, UK) as secondary antibodies for 60 min at room temperature. The sections were observed and photographed under a fluorescence microscope (NIKON Eclipse Ci-L, Japan). Th⁺ immunofluorescence staining analyzed the mean number of nerve fibers in five fields randomly was quantified and plotted as per mm² (45 (link)).