Mac3 clone m3 84

Immunohistochemistry was performed as described before [44 (link),45 (link)]. Mice were sacrificed under deep anesthesia by intra-cardiac perfusion with PBS followed by perfusion with 4% (w/v) PFA dissolved in PBS. Spinal cords and brains were removed and fixed in 4% PFA overnight. Before embedding in paraffin and sectioning in 3 µm slices, the spinal cord was cut into 7–10 transverse segments (3 mm thick) and coronal brain cuts were made. The sections were pretreated with citrate buffer (pH 6 or 9) in a steamer. Immunohistochemistry was then performed using the biotin–streptavidin peroxidase technique (Dako by Agilent Technologies, Santa Clara, CA, USA) with an automated immunostainer (AutostainerLink 48, Dako by Agilent Technologies, Santa Clara, CA, USA). The primary antibodies used were specific to Mac3 (clone M3/84; BD Pharmingen, San Jose, CA, USA) or CD3 (MCA, Serotec, Oxford, UK). 3,3′-diaminobenzidine (Dako by Agilent Technologies, Santa Clara, CA, USA) was used as color substrate and sections were mounted with Eukitt^® mounting medium (O. Kindler GmbH, Bobingen, Germany) after dehydration. Images were acquired with the microscope AxioObserver (Carl Zeiss, Jena, Germany) or BioRevo BZ-9000 (Keyence, Itasca, IL, USA) using the software AxioVision or BZ-II Analyzer (Keyence, Itasca, IL, USA).

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.