Tissues labeled with anti-MBP continue with a secondary Ab labeling step consisting of 1 h incubation with biotinylated IgG Ab at 1:1000 dilutions (Vector Labs), www.selleckchem.com/products/avelestat-azd9668.html followed by 1.5-h incubation with strepavidin Ab conjugated to Alexa 647 fluorochrome (Chemicon). All other tissues follow with secondary Ab conjugated to TRITC or Cy5 (Vector Labs and Chemicon) for 1.5 h. To assess the number of cells, a nuclear stain DAPI (2 ng/mL; Molecular Probes) was added 10 min prior to final washes after secondary Ab incubation. Sections were mounted on slides, allowed to semi-dry, and cover slipped in fluoromount G (Fisher Scientific). IgG-control experiments were performed for all primary Ab, and only non-immunoreactive tissues under

these conditions were analyzed. Stained sections were examined and photographed using a confocal microscope (Leica TCS-SP, Mannheim, Germany) or a fluorescence microscope (BX51WI; Olympus, Tokyo, Japan) equipped with Plan Fluor objectives connected to a camera (DP70; Olympus). Digital images were collected and analyzed using Leica confocal and DP70 camera software. Images were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). To quantify immunohistochemical staining results, three spinal cord cross-sections PF-02341066 ic50 at the T1–T5 level from each mouse (n=3) were captured under microscope at 10× or 40× magnification using the DP70

Image software and a DP70 camera (both from Olympus). All images in each experimental set were captured under the same

light intensity and exposure limits. Image analysis was performed using ImageJ Software v1.30, downloaded from the NIH website (http://rsb.info.nih.gov/ij). Axonal densities were calculated by counting the number of NF200+ cells in a 40× image over the area of the captured tissue section. Inflammatory infiltrates were quantified by measuring the intensity of CD45 staining in captured 10× images. EAE severity significance was determined by repeated measures one-way ANOVA. Immunohistochemical and flow cytometry data were analyzed by bootstrap one-way ANOVA and paired t-test, respectively. For these analyses, the mean or median was used as the comparator, and the F-stat equation was modified such that absolute values replaced the squaring of values. For bootstrap one-way ANOVA, post hoc analysis was performed on F-stat values and Osimertinib ic50 significance was determined at the 95% confidence interval. The authors acknowledge Tina Chung, BS for technical laboratory assistance and Stefan Gold, PhD for helpful suggestions and discussions. The support for this work was provided by National Institutes of Health grant K24NS062117 and National Multiple Sclerosis Society grants RG 3593, 4033, and CA1028 to R.R.V., as well as by the Skirball Foundation, the Hilton Foundation, and the Sherak Family Foundation. Conflict of Interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”.