Spinal Cord Motor Neuron Analysis

The L1–L4 lumbar level of the spinal cords were cut on a vibratome into coronal sections of 40 μm. Four nonadjacent sections spaced by 320 μm were labelled by immunohistochemistry as previously described [25 (link)], using a goat anti-choline acetyltransferase (ChAT) antibody (Millipore, Burlington, MA, USA) and a biotinylated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA). Two images per section (one per ventral horn) were captured using an AxioImager. M2 microscope (Zeiss, Oberkochen, Germany) equipped with a high-resolution B/W camera (Hamamatsu Photonics, Hamamatsu City, Japan) and run by the ZEN 2 software (Zeiss). Cell body sizes were measured using ImageJ (NIH, Bethesda, MD, USA).