Rabbit anti mtor antibody

Immunohistochemical staining was performed on fixed brain or spinal cord. Animals were killed 7 days after intratheral delivery of IL-6, IL-6 and AG490 or saline (n = 4 respectively), by transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS). The brain and 10 mm spinal cord centered at the lesion site were dissected, postfixed in the same fixatives, and soaked in 30% sucrose solution. 30 μm thickness of cryostat sections of the brain through the sensorimotor cortex or the spinal cord were cut in coronary section. Sections were blocked in TBS with 5% normal donkey and 5% normal goat serum for 1 h and then incubated with primary antibodies overnight at 4°C. Fluorescence conjugated secondary antibodies were applied for 2 h at room temperature. The primary antibodies were omitted in the primary antibody negative control group. The following primary antibodies were used: mouse anti-βIII tubulin antibody (1:400, Sigma), rabbit anti-gp130 (1:200, Bioworld), rabbit anti-pSTAT3 (S727) antibody (1:100; Bioworld), rabbit anti-mTOR antibody (1:500; Cell Signaling). Images were analyzed with Image J software to measure the fluorescence intensity by drawing around cell bodies of mTOR or pSTAT3 positive neurons after deduction the background signal. Each group 100 neurons were analyzed.