Histological Analysis of Demyelination and Inflammation

Mice were euthanized under deep anesthesia (ketamine 180 mg/kg; xylazine 16 mg/kg) by intracardiac perfusion with phosphate-buffered saline (PBS) followed by perfusion with 4% (w/v) paraformaldehyde (PFA) solved in PBS. Brain and spinal cord were removed and fixed in 4% PFA overnight. Prior to paraffin embedding, the spinal cord was cut into seven to ten transverse segments (3 mm thick) and coronal brain cuts were made. Sections (3 μm) were stained by HE (hematoxylin and eosin) and LFB-PAS (luxol fast blue including periodic acid-Schiff). Immunohistochemistry was performed using a biotin-streptavidin peroxidase technique (Dako) and an automated immunostainer (AutostainerLink 48, Dako). Sections were pretreated with citrate buffer (pH 6) in a steamer for immunohistochemistry for Mac3 (clone M3/84, #550292, 1:200 also known as CD107b or LAMP-2; BD Pharmingen). Quantification of demyelination and Mac3 infiltration was performed as described previously^{67 (link),68 (link)}.