Transverse sections (40 μm thick) were obtained with a cryostat (Leica, Heidelberg, Germany) individually placed on 96-well plates in Olmos solution and stored at −20°C. The sections were distributed in 50 series of five sections each, and each series was prepared for immunohistochemical analysis by blocking with 10% bovine serum, 0.3% Triton X-100 in tris-buffered saline for 1 h at room temperature, followed by incubation

with different combinations of up to three primary antibodies against synaptotagmin (clone Mab48, Developmental Studies Hybridoma Bank, IA), nitrotyrosine (Millipore, Bedford, MA), human HCA-ABC antigen (MHC-I, DAKO, Glostrup, #selleck chemical keyword# Denmark), MHC-II-APC (eBiosciences, SanDiego, CA), Iba1 (Wako, Tokyo, Osaka, Japan), sigma 1 Receptor (Sig1-R, Santa Cruz Biotechnologies, Santa Cruz, CA) and ChAT (Millipore) Inhibitors,research,lifescience,medical overnight at 4°C. After washes, sections were incubated for 1 day at 4°C with biotinylated secondary antibodies (Vector, Burlingame, CA, 1: 200) with Cy-2, Cy-3, or Cy-5 conjugated donkey anti-rabbit, anti-mouse, or anti-goat IgGs antibodies

(Jackson Immunoresearch, Inhibitors,research,lifescience,medical West Grove, PA, 1:200). Slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma, St Louis, MO, 1: 1000) and mounted with Fluoromount (SouthernBiotech, Birmingham, AL). Omission of the primary antibodies resulted in no detectable staining. At lumbar levels, the analysis was focused in MNs from L4–L5 segments that provide innervation to hindlimb muscles. Sections from different time points of transgenic and Inhibitors,research,lifescience,medical control animals were processed in parallel for immunohistochemistry and data represent an accumulation of different day performances. Confocal microscope Inhibitors,research,lifescience,medical examinations were performed with a Leica TCS SP2 AOBS laser scanning confocal system (Leica). All MNs were analyzed in a z-plane containing the nucleus and captured using the FV10-ASW 1.7 Viewer software. Confocal images were obtained

using two separate photomultiplier channels, either concurrently or in separate runs, and were separately projected and merged using a pseudocolor display showing green for Cy2, red for Cy3, magenta for Cy5, and blue for DAPI. When densitometric analysis was performed, images of the ventral area of the spinal cord were taken under the same exposure time, sensibility, and resolution for each marker analyzed, DNA ligase with the aid of a digital camera (Olympus DP50) attached to the microscope (Olympus BX51). The microphotographs were transformed to a gray scale and analyzed using ImageJ software. Immunoreactivity was assessed by calculating the integrated density, after defining a threshold for background correction. The integrated density of a region of interest (ROI), defined as the area above the threshold for the mean density minus the background, was measured.