PCR products were separated on a 1·5% agarose gel and analysed by Image Pro-Plus software (Media Cybernetics, Silver Springs, MD, USA). Real-time

PCR was performed by an ABI STEPONE real-time PCR system using the SYBR Green real-time PCR kit (Roche Ltd, Basel, Switzerland). The primers used to amplify IFN-γ [38] (5′-GATGCATTCATGAGTATTGCCAAGT-3′, 5′-GTGGACCACGCGGATGAGCTC-3′), IL-27 p28 [39] (5′-TTCCCAATGTTTCCCTGACTTT-3′, 5′-AAGTGTGGTAGCGAGGAAGCA-3′), IL-27 EBI3 [39] (5′-TGAAACAGCTCTCGTGGCTCTA-3′, 5′-GCCACGGGATACCGAGAA-3′) and MHC-II [40] (5′-GCGACGTGGGCGAGTACC-3′, 5′-CATTCCGGAACCAGCGCA-3′) were used to detect MI-503 in vitro the expression of respective genes. The data were normalized against GAPDH (5′-CGGCCGCATCTTCTTGTGCA-3′,

5′-GCCGTGAGTGAGTCATACT-3′) levels. The amplification of real-time PCR was performed with an initial denaturation of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative gene expression levels were quantified using the comparative ΔCT method. This method normalized CT values of the detected gene to the average of that of the GAPDH and calculated the relative expression values as fold changes of the control, which was set at 1. Melting curve analyses and electrophoresis were performed to verify the specificity of the PCR products. Frozen spinal cord sections were dually stained with goat anti-mouse GFAP (Santa Cruz Laboratories, Santa ABT 888 Cruz, CA, USA) and rat anti-mouse MHC-II (Santa Cruz Laboratories), followed by incubation with fluorescein isothiocyanate (FITC)-labelled anti-rat and tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)-labelled anti-goat secondary antibodies (ZSGB-Bio, Galeterone Beijing, China). Stained sections were examined and photographed using fluorescence microscopy (Carl Zeiss, Germany) and scanning confocal laser microscopy (Leica, China). Astrocytes were treated with or without 100 U/ml IFN-γ and then co-cultured with lymphocytes obtained from lymph node at a lymphocyte : astrocyte ratio

of 10:1 for 72 h. Twenty-five μg/ml MOG35–55 peptide was incubated in the culture as antigen. Astrocytes were lysed in lysis buffer containing protease inhibitors, and cell lysates were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane via semidry transfer. Membranes were blocked with 5% non-fat milk for 1 h at room temperature and IL-27 (Santa Cruz, CA, USA) expression was detected. All antibodies were diluted with Tris-buffered saline with 0·1% Tween 20 (TBST). GAPDH was used as reference genes. The optical density of bands was evaluated using Scion Image Beta version 4·02 (Scion Corporation, Frederick, MD, USA) and statistical comparison was performed with GraphPad Prism version 5 software. Data are expressed as means ± standard error of the mean (s.e.m.).