Cd3 clone cd3 12

For histology, mice were intracardially perfused with 20 ml cold PBS and fixed by perfusion with 10 ml of 4 % paraformaldehyde (PFA). Spinal cords were removed and kept in PFA for 48 h at 4 °C. The fixed spinal cords were cut into 3 mm thick transverse segments and embedded in paraffin. To evaluate demyelination, spinal cord sections were stained with Luxol Fast Blue and subsequently incubated with Periodic acid-Schiff. Immunohistochemistry was performed using the biotin-streptavidin peroxidase technique (K5001, Dako) in an immunostainer (AutostainerLink 48, Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or pH 9.0 (Dako)) before incubation with the primary antibodies against CD3 (clone CD3-12, BioRad, 1:100) or Mac3 (clone M3/84, BD, 1:100) or B220 (clone RA3-6B2, BD, 1:200). DAB (3,3ʼ-Diaminobenzidin) was used as a chromogen. For B220/Ki67 double-immunofluorescence staining, B220 (clone RA3-6B2, BD, 1:100) and Ki67 (clone SP6, Thermo Scientific, 1:100) were used as primary antibodies; AF488- and AF594-labeled secondary antibodies (both 1:100) were used for visualization. Stained sections were analysed with a keyence microscope and pictures were taken with an Axioplot camera. ImageJ v1.48 was used to manually count infiltrated cells and measure areas.