ntibodies and reagents Two hybridoma strains for the mouse anti-rat NogoA protein were preserved by the Institute of Neurosciences in the Fourth Military Medical University, and the mouse IgG was purified as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645759 described previously. We purchased the following primary antibodies: polyclonal rabbit anti-NogoA antibody , rabbit anti-MBP mAb, rabbit anti-GFAP mAb, rabbit anti-GST, anti-Tau, anti-Map2, anti-bIIItubulin, and anti-b-actin. The following secondary antibodies were used: -labelled goat anti-mouse immunoglobulin, Alexa-594-labelled goat antirabbit IgG, and hydrogen peroxidase conjugated goat anti-rabbit and anti-mouse IgG. Recombinant Rat NogoA/Fc Chimera and Recombinant Rat NogoA/Fc Chimera were purchased from R&D Systems. Western blot and IHC staining length thoracolumbar segment of the spinal cord was removed and put into 25% sucrose in 0.1 M phosphate buffer for 36 h at 4uC. Serial coronal sections of a 12 mm thickness were prepared using a freezing microtome. The sections were post-fixed in 4% PFA for 1 h at room temperature. Subsequently, the sections were rinsed with 0.01 M phosphate-buffered saline and then blocked with 1% BSA in PBS containing 0.3% Triton X-100 for 1 h at room temperature. The sections were divided into six groups for the different primary antibodies: I, aNogo66 mAb and Anti-NogoA pAb; II, aNogoA-N mAb and Anti-NogoA pAb; III, aNogo66 mAb and anti-MBP mAb; IV, aNogoA-N mAb and anti-MBP mAb; V, aNogo66 mAb and anti-GFAP mAb; VI, aNogoA-N mAb and anti-GFAP mAb. All sections were MedChemExpress Vesnarinone incubated in primary antibody at 4uC for 24 h. After washing with PBS three times, the secondary antibodies were incubated in a dark environment at room temperature for 2 h. The stained sections were then washed with PBS three times and mounted with glycerol. The sections were observed under an Olympus BX-51 microscope. Expression and purification of recombinant proteins First, we cloned two fragment sequences from the Nogo66 truncation and four fragment sequences from the NogoA N-terminal truncation by reverse transcriptase-polymerase chain reaction. Then, the PCR product was subcloned into the EcoR I and Sal I sites of pGEX4T-1. After sequencing, all plasmids containing the truncated fragments were transformed into BL21 E. coli for expression. The recombinants were GST-DNogoA-Na, 43 kDa; GST-DNogoA-Nb, 38 kDa; GST-DNogoA-Nc, 33 kDa; GST-DNogoA-Nd, 30 kDa; GST-DNogo66a, 33 kDa; GST-DNogo66b, 30 kDa. The GST protein is 26 kDa and was expressed from the empty pGEX4T-1 vector. In this study, the expression of recombinant proteins was under the control of the Tac promoter and was induced by isopropyl-b-D-thiogalactopyranoside. After 2 h of shaking, a final concentration of 0.5 mM IPTG was added into the Luria-broth medium with ampicillin, and the medium was further agitated for 3 h. Subsequently, the bacterial cells were pelleted at 12000 g for 6 min, buffer was added, and the cells were boiled for 10 min. SDS-PAGE was performed to analyse the molecular size of the recombinant proteins using Coomassie blue staining and WB analysis. WB was performed briefly as follows. The antibodies were diluted as follows: rabbit anti-GST antibody was diluted 1:30000 in blocking solution and incubated with the blots for 2 h at room temperature; the secondary antibodies were Antibodies of NogoA Enhance Axon Extension -conjugated goat anti-rabbit IgG and were incubated with the blots for 1 h at room temperature. Antibody binding was visualised using an enh