The sciatic nerves were fixed in 4% paraformaldehyde for immunohistochemistry and then embedded in paraffin according to a published protocol [13 (link)]. Three cross sections (6-μm-thick) or three longitudinal sections (6-μm-thick) at 60 μm apart per animal were used [13 (link)].
Epidermal foot pads from left hind feet were fixed in Zamboni's fixative for 2 hours, washed in PBS, and then kept in 30% sucrose/PBS overnight at 4°C. The samples were embedded in OCT compound and stored at −80°C. Three longitudinal 20-μm-thick footpad sections from each mouse were prepared.
The following primary antibodies were used: polyclonal rabbit anti-myelin basic protein (MBP, 1 : 400, Dako Denmark, USA), polyclonal antineurofilament, heavy chain (NF-H, 1 : 1000, Thermo Scientific, USA), polyclonal rabbit anti-Ang1 (1 : 2000; Abcam, USA), polyclonal rabbit anti-S100 (1 : 400, Abcam, USA), and polyclonal rabbit anti-protein gene product 9.5 (PGP 9.5, 1 : 1,000; MILLIPORE, USA). Rabbit or goat IgG was used as a negative control. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 : 5000, Thermo Scientific, USA).
Epidermal foot pads from left hind feet were fixed in Zamboni's fixative for 2 hours, washed in PBS, and then kept in 30% sucrose/PBS overnight at 4°C. The samples were embedded in OCT compound and stored at −80°C. Three longitudinal 20-μm-thick footpad sections from each mouse were prepared.
The following primary antibodies were used: polyclonal rabbit anti-myelin basic protein (MBP, 1 : 400, Dako Denmark, USA), polyclonal antineurofilament, heavy chain (NF-H, 1 : 1000, Thermo Scientific, USA), polyclonal rabbit anti-Ang1 (1 : 2000; Abcam, USA), polyclonal rabbit anti-S100 (1 : 400, Abcam, USA), and polyclonal rabbit anti-protein gene product 9.5 (PGP 9.5, 1 : 1,000; MILLIPORE, USA). Rabbit or goat IgG was used as a negative control. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 : 5000, Thermo Scientific, USA).
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