Mice were deeply anaesthetized using an overdose of pentobarbital (200 mg/ml) containing lidocaine (20 mg/ml) and perfused through the left ventricle with cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS).
Toluidine blue (TB) staining and electron microscopy (EM) analyses: For EM and TB analyses of axons and myelin, 1 mm segments of the thoracic spinal cords were processed as previously described [9 ]. One µm thick sections were obtained with a Leica Ultracut E microtome and stained with 1% TB solution.
Paraffin histopathology and immunohistochemical analysis: Spinal cords were removed and tissue segments containing the lesion area were paraffin-embedded and cut into 10 parallel series of 15µm thick microtome sections. Furthermore, in naïve mice lung, heart, liver, diaphragm, spleen, the anterior tibialis muscle and small intestine biopsies were imbedded in paraffin and cut into 5µm thick sections. Sections were then stained using Hematoxylin and Eosin (H&E) [3 (link)]. For stereological analysis of the total number of astrocytes in the hilus of the hippocampus, brains were processed as previously described [18 (link)] and cut horizontally into six 60µm-thick parallel series of free floating vibratome sections and cryo-protected in de Olmo’s solution.
Toluidine blue (TB) staining and electron microscopy (EM) analyses: For EM and TB analyses of axons and myelin, 1 mm segments of the thoracic spinal cords were processed as previously described [9 ]. One µm thick sections were obtained with a Leica Ultracut E microtome and stained with 1% TB solution.
Paraffin histopathology and immunohistochemical analysis: Spinal cords were removed and tissue segments containing the lesion area were paraffin-embedded and cut into 10 parallel series of 15µm thick microtome sections. Furthermore, in naïve mice lung, heart, liver, diaphragm, spleen, the anterior tibialis muscle and small intestine biopsies were imbedded in paraffin and cut into 5µm thick sections. Sections were then stained using Hematoxylin and Eosin (H&E) [3 (link)]. For stereological analysis of the total number of astrocytes in the hilus of the hippocampus, brains were processed as previously described [18 (link)] and cut horizontally into six 60µm-thick parallel series of free floating vibratome sections and cryo-protected in de Olmo’s solution.
