In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

The remaining tissue was processed for imm~hemistry of I #m frozen sections following the Tokuyasu technique (1973 Tokuyasu technique ( , 1980 . Transverse slices of spinal cord 0.5--0.7 mm thick were posttixed overnight at 4°C in the aldehyde fixative solution described above. Afterwards the blocks were immersed sequentially for 12 h each in 5%, 15%, 50%, and 2.3 M sucrose in PBS at 4°C. In some eases, 30% polyvinylpyrrolidone (molecular mass 10 kD; Sigma Chemical Co., St. Louis, MD) was added to the 2.3-M sucrose solution; this improved the cutting properties of the blocks and reduced artifactual separation of tissue elements within the white matter. Nevertheless, some separation of elements was unavoidable in actively demyelinating specimens with abundant vacuolization. After sucrose infiltration, each piece was trimmed to an appropriate size, mounted on a specimen holder, and frozen by immersion in liquid nitrogen. The specimens were stored in liquid nitrogen until use. Transverse sections 1 pm thick were cut from those blocks using glass knives and a Reichert-Jung Ultracut E microtome with ultracr~tomy system FC4D. The sections included in most cases at least a complete half face, from the midline to the lateral edge of the spinal cord. Sections were transferred on 2.3 M sucrose drops to slides coated with a dried aqueous solution of gelatin (1% pig skin gelatin; Sigma Chemical Co.) and chromium potassium sulfate (0.1%) in PBS. Slides bearing sections were then immersed in 4% formaldehyde in PBS for 5 rain and stored in PBS at 4°C for 1-2 d before processing for immunocytoehemistry.