Immunohistochemical Staining of Spinal Cord and Brain

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).