The lumbar spinal cord was dissected, embedded in paraffin and serial sections were obtained. Of every 7 parts, the first, fourth and seventh were stained with cresyl violet. The fifth and sixth were immunoreacted for Bax or Bcl 2, respectively. The third was employed for TUNEL labeling. Twenty two parts were discarded after Crizotinib structure every 7section series. After cresyl violet staining, motoneurons of the group of the lumbar enlargement were measured. Neuronal counting was done blind to treatment. Only significant multipolar motoneurons with a plainly visible nucleolus were considered. In animals, the unoperated contralateral side of the spinal cord was used as control. The ratio between the number of motoneurons counted in-the control and operated sides was understood to be motoneuron survival ratio. For immunostaining techniques, endogenous peroxidase was blocked with three full minutes hydrogen peroxide/10 mM PBS pH 6. 0 for 15 min. A while later, the sections were microwaved in 10 mM citrate buffer pH 6. 0 and incubated overnight with primary antibody both to Bax or Bcl 2 and sc 492, respectively) applied in 1:200 dilution at 20 C. The sections were incubated with biotinylated secondary antibody, included with peroxidase conjugated streptavidin solution and prepared Endosymbiotic theory for DAB reaction. Any cell form demonstrating cytoplasmic staining was considered good for qualitative research. TUNEL reaction was conducted using the TdT FragELTM DNA Fragmentation Detection Kit according to the manufacturers directions. All steps were done at 20 C, except TdT chemical reaction. In temporary, sections were deparaffinized and permeabilized with proteinase K/10 mM Tris pH 8. 0. Endogenous peroxidase was blocked with three years H2O2/methanol. The sections were incubated with equilibration buffer then with TdT enzyme solution and after with stop buffer. Described DNA was discovered by applying blocking load followed by peroxidase conjugated streptavidin option and DAB reaction. Methyl green was used as counterstain. For each animal, six sections were used for counting the little Bax or TUNEL positive cells inside the GW0742 side ipsilateral to the axotomy. Sections were examined with the aid of an atlas to identify the parts which roughly match Rexed laminae I?VI and IX of the lumbar level. At 1, 3 or 5 days postaxotomy, the mice were anesthetized by hypothermia and, after immediate dissection of the lumbar enlargement, were decapitated. Total RNA in the lumbar enlargement was separated through the use of TRIzol chloroform, reagent and isopropyl alcohol following manufacturers protocol.