The wounded skin was excised and fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 24 h to prevent cell autolysis after death. Then, the sections were hydrated with running water for a certain period of time, followed by gradual dehydration with alcohol at different concentrations, embedding in paraffin and cutting into 5 μm sections. The sections were stained with hematoxylin-eosin (HE), and the histological changes of the wounds were visualized under a microscope.
Skin tissues were sectioned into 5 μm sections as above. For immunofluorescence double staining, sections were incubated overnight at 4°C with a mix of primary anti-PGP9.5 (GB11159-1, 1:1,000, Servicebio) and anti-GAP43 (bs-0154R, 1:4,000, Bioss) antibodies. Sections were incubated in Cy3 conjugated Goat Anti-Rabbit IgG (H+L) (GB21303, 1: 300, Servicebio) or HRP conjugated Goat Anti-Rabbit IgG (H+L) (GB23303, 1:500, Servicebio) secondary antibody for 1 h at room temperature. Images were captured by laser scanning confocal microscope (Olympus, Japan), and the confocal software was used for acquisition of the data and merging of the digital images. Each antibody was validated separately prior to use in double immunofluorescence.
Skin tissues were sectioned into 5 μm sections as above. For immunofluorescence double staining, sections were incubated overnight at 4°C with a mix of primary anti-PGP9.5 (GB11159-1, 1:1,000, Servicebio) and anti-GAP43 (bs-0154R, 1:4,000, Bioss) antibodies. Sections were incubated in Cy3 conjugated Goat Anti-Rabbit IgG (H+L) (GB21303, 1: 300, Servicebio) or HRP conjugated Goat Anti-Rabbit IgG (H+L) (GB23303, 1:500, Servicebio) secondary antibody for 1 h at room temperature. Images were captured by laser scanning confocal microscope (Olympus, Japan), and the confocal software was used for acquisition of the data and merging of the digital images. Each antibody was validated separately prior to use in double immunofluorescence.