Viral Infection of Transgenic Mice Expressing a Viral Protein in Oligodendrocytes Leads to Chronic Central Nervous System Autoimmune Disease

Evans, Claire F.; Horwitz, Marc S.; Hobbs, Monte V.; Oldstone, Michael B.A.

Immunohistochemical Staining of CNS Tissues. Mice were anesthetized and perfused with 40 ml saline. Brains and spinal cord were removed, covered with Tissue-Tec OCT (Miles Diagnostics Division, Elkhart, IN), snap-frozen at Ϫ 80 Њ C in isopentane, and then stored at Ϫ 20 Њ C. Immunohistochemistry was performed on 10-12-m cryostat sections that were fixed in 100% ethanol for 15 min at 4 Њ C and blocked with avidin and biotin (Vector Laboratories, Burlingame, CA). Staining was done with the following primary antibodies: anti-CD8 (anti-Ly-2 and Ly-3, Phar-Mingen, San Diego, CA), anti-CD4 (anti L3T4; PharMingen), anti-H-2 monotypic antigen (MHC class I), anti-Ia antigen (MHC class II), anti-B220 (Boehringer Mannheim, Indianapolis, IN), and anti-F4/80 (Serotec, Oxford, England). The second antibody was a biotinylated anti-mouse IgG and was used in conjunction with the Vectastain Elite ABC (peroxidase) kit (Vector Laboratories). Staining was detected using diaminobenzidine as a chromagen. Sections were counterstained in Mayer's hematoxylin (Sigma, St. Louis, MO) and mounted in Aqua-Mount (Lerner Laboratories, Pittsburgh, PA).