Anti mouse and anti rabbit secondary antibodies

Spinal cord sections from EAE models (at 19 dpi, n = 6 in each group) were stained with neurofilament (NF) mouse mAb (Cell Signaling Technology, Beverly, MA, USA) followed by anti-mouse secondary antibody (Abcam (Hong Kong) Ltd, New Territories, HK) for evaluating axonal damage. Moreover, spinal cord sections and astrocytes cultured in vitro were dually stained with mouse anti-mouse GFAP (Millipore, Bedford, MA, USA) and rabbit anti-mouse CCL20 (Abcam (Hong Kong) Ltd, New Territories, HK), followed by incubation with anti-mouse and anti-rabbit secondary antibodies (Abcam (Hong Kong) Ltd, New Territories, HK). Stained sections were examined and photographed using a Leica DMI 4000B microscope (Leica Corp., Lasertechnik, Heidelberg, Germany) (for spinal cord sections) or Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) (for astrocytic cultures). Mean fluorescence intensity was calculated using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA).

Cells were resuspended in lysis buffer and incubated on ice for 30 min. The cell suspension was homogenized and then centrifuged at 12,000 r.p.m. for 10 min at 4 °C. The supernatant was transferred to a new tube and denatured at 95 °C for 10 min before loading. Equal amounts of protein were separated in 10% SDS-polyacrylamide gel and then transferred to PVDF membranes (GE Healthcare). The primary antibodies used in this study included: anti-GAPDH antibody and anti-Sox2 antibody from Cell Signaling Technology, anti-PAI-1 antibody from Santa Cruz. Both anti-mouse and anti-rabbit secondary antibodies were from Abcam.