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

The absence of cross-reaction between the three antibody channels was tested by omitting in turn each one of the three primary antibodies while leaving the other two in the primaries mixture and while including all three secondaries in the secondaries mixture. Omission of each primary completely abolished binding of the corresponding secondary, while signals in the other two channels remained unchanged. In addition, each section provided its own further demonstration of the specificity of the three channels, since each of three primaries exhibited unique and characteristic regionai and intracellular patterns of staining. Identification of all double or triple labeled cells was not possible. We identified cells as multiply labeled only when we saw coincident, overlapping, or concentric multiple labeling and when the corresponding phase contrast image was consistent with that interpretation,