Goat anti mouse antibodies coupled

Cryostat sections were exposed to 5% normal goat serum in 0.01 M PBS with 0.03% Triton X-100 at room temperature for 1 h prior to the primary antibody incubation. Nerve sections were incubated for 1 h at room temperature followed by overnight incubation at 4 °C with rabbit polyclonal antibodies against high molecular weight neurofilament (NF; 1:250, Millipore AB1991) to stain axons, mouse monoclonal antibodies against S100 (1:200, Millipore MAB079–1) combined with rabbit polyclonal antibodies against P75 (1:100, Millipore 07–476) to stain Schwann cells. After the primary incubation, nerve tissue sections were washed three times for 5 min with 0.01 M PBS (pH 7.4) and then incubated with goat anti-rabbit or goat anti-mouse antibodies coupled to AlexaFluor 488 or AlexaFluor 555 (1:500; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Sections were then washed 3 times for 5 min with 0.01 M PBS (pH 7.4), counterstained with DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride; ThemoFisher), rinsed twice more, and covered with glass slips with anti-fade fluorescent mounting medium (Dako, Carpenteria, CA, USA). For detection of myelinated axons, plastic sections were stained with toluidine blue, covered with glass slips with VectaMount mounting medium (Vector Laboratories, Burlingame, CA, USA).