Immunocytochemistry Protocols for Oligodendrocyte Markers

For immunocytochemistry, the cells were fixed with 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min at room temperature; washed with PBS; and blocked for 45 min at room temperature with blocking solution (PBS containing 5% normal donkey serum, 0.5% Triton X-100 and 0.05% sodium azide). The samples were incubated overnight at 4°C with primary antibodies diluted in blocking solution, washed three times in PBS, incubated for 45 min with secondary antibodies and washed in PBS and mounted with mounting medium (Vectashield H-1400, vectorlabs). For primary antibodies, the following antibodies were used: rat monoclonal antibody to MBP (1 : 300, MAB386, Chemicon), mouse hybridoma supernatants to O4 (1 : 5), rabbit polyclonal antibodies to Olig2 (1 : 500, AB9610, Chemicon) and Caspr (1 : 1000) [22 (link)]. Secondary antibodies were obtained from Jackson Immunoresearch. DAPI was obtained from Invitrogen.