Quantification of RGC density in various experimental groups

ed to 45 uL of blank control plasma or blank spinal cord homogenate in a 96 deep-well plate. One Vonoprazan price hundred fifty microliters of acetonitrile containing 500 ng/mL of pyrimethamine PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723632 as an internal standard and 0.1% formic acid were added to each well. The plate was vortexed vigorously and then centrifuged at 500xg for 30 minutes at 4 degrees C. One hundred microliters of the supernatant were pipetted into a new 96 well plate for LC-MS/MS analysis. Plasma and spinal cord homogenate samples from mice assigned to the guanabenz pk study were handled similarly without guanabenz spiking 11 / 15 Guanabenz Treatment Accelerates Disease in a SOD1 Mouse Model of ALS steps. Quantitation of guanabenz in tissues from the mice was interpolated by comparison of the signal to that generated by the standard curve. HPLC and mass spectrometer conditions for guanabenz were the same as those reported for detection of dexpramipexole in a previous report. Quantitative PCR Tissue samples were homogenized in 1 mL Tri Reagent by Precellys 24 homogenizer for two 20 second intervals. Next 0.1 mL of bromochloropropane was added the homogenate and mixed. After 10 minutes, samples were centrifuged at 12,000 x g for 15 minutes. RNA purification was carried out using Agencourt RNAdvanced kit on Biomek Laboratory Automation Workstation. RNA samples were than used to generate cDNA samples using Applied Biosystems High Capacity cDNA reverse transcription kits. Quantitative PCR reactions were carried out using Applied Biosystems 7900HT Fast Real-Time PCR System. Target gene probe sets used were Atf4, Bcl2, Bip, Ccnd1, and Chop. Endogenous control probe sets used were Gapdh, Canx, and Ubc. Relative gene expression was determined using the GeNorm expression normalization algorithm. Transformed relative expression data were then compared using GraphPad Prism software one-way analysis of variance and Tukey’s post-hoc test. Western Blots Western blots were carried out using the following primary rabbit monoclonal antibodies from Cell Signaling, Inc: Bcl-2 rabbit mAb, BiP rabbit mAb, Chop rabbit mAb, Cyclin D1. All of the above antibodies were incubated overnight at 1:1000 dilutions. A western blot was also carried out for detection of Gapdh using a mouse monoclonal antibody from Santa Cruz Biotechnology. Relative levels of Chop, Bcl2, and Bip proteins were compared by calculating fluorescent signal on western blots using the Leicor Odyssey Infrared Scanner. Chop, Bcl2, and Bip were all normalized with Gapdh as a loading control. Relative values of each target protein from treated mice were compared to vehicle control using t-test. Mouse Survival Efficacy Testing We used general survival efficacy testing methods previously described. for the current studies. For both efficacy studies, transgene copy number was verified and mice were assigned to either drug treatment or vehicle treatment groups at 50 days of age. Groups were balanced with respect to gender and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19723666 body weight within gender. Litters were evenly represented across treatment and vehicle groups. Observers were blinded to treatment groups. For the intraperitoneal dosing study, vehicle was a 5% glucose solution prepared by diluting a 45% glucose stock solution with water. For the treated cohort, 5.06 mg of guanabenz acetate salt was diluted into 10 mL of 5% glucose solution for a 0.4 mg/ml guanabenz free base solution on each day of treatment administration. Intraperitoneal injection volumes for both vehicle and guanabenz treat