A phosphorylated S6 (pS6)‐specific antibody (S235/236, Cell Signaling, Danvers, MA, USA) was used to monitor mTORC1 activation. Antibodies for neuronal nuclei (NeuN; Chemicon, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; Dako), neuron‐glial antigen 2 (NG2; Millipore, Billerica, MA, USA), OX‐42 (Serotec Oxford, UK), ED1 (Serotec Oxford, UK), Ki‐67 (BD Pharmingen Oxford, UK), and myeloperoxidase (MPO; Hycult Biotech, Uden, the Netherlands) were used to evaluate neurons, astrocytes, oligodendrocyte progenitors, microglia/macrophages, activated microglia/macrophages, proliferation, and neutrophils, respectively. Primary antibodies were visualized using appropriate secondary antibodies (Cy3 or DyLight™ 488, Jackson Laboratories, Sacramento, CA, USA).
Spinal cord sections (20 μm) mounted on gelatin‐coated slides were incubated with primary antibody at 4°C for 24 h. After washing, secondary antibodies were added and sections incubated for 1.5 h at room temperature. Primary and secondary antibodies were diluted in 0.3% Triton‐X 100 in 0.1 M PBS. Slides were coated with an anti‐fade agent (Prolonged Gold® Invitrogen, Carlsbad, CA, USA) and were visualized using fluorescence microscopy (Nikon Eclipse TE3000, Surrey, UK) if only labeled with one marker or confocal microscopy (Olympus FV1000 CLSM, Hamburg, Germany) if double‐labeled.
Spinal cord sections (20 μm) mounted on gelatin‐coated slides were incubated with primary antibody at 4°C for 24 h. After washing, secondary antibodies were added and sections incubated for 1.5 h at room temperature. Primary and secondary antibodies were diluted in 0.3% Triton‐X 100 in 0.1 M PBS. Slides were coated with an anti‐fade agent (Prolonged Gold® Invitrogen, Carlsbad, CA, USA) and were visualized using fluorescence microscopy (Nikon Eclipse TE3000, Surrey, UK) if only labeled with one marker or confocal microscopy (Olympus FV1000 CLSM, Hamburg, Germany) if double‐labeled.