The brains, spinal cords, and optic nerves were removed from mice and fixed in 10% neutral-buffered formalin, paraffin-embedded, and sectioned as described previously.5 (link),15 (link) Representative sections were stained with Luxol fast blue–hematoxylin & eosin and reticulin preparation (for connective tissue). Stained tissue specimens were examined by light microscopy. A blinded observer (R.A. Sobel) counted both meningeal and parenchymal inflammatory foci (>10 clustered inflammatory cells). Avidin-biotin immunohistochemical staining was performed on the sections with rabbit anti-mouse CD3 (Abcam, Cambridge, UK) and rat anti-mouse CD45R (B220; BD Biosciences) using reagents from Vector Laboratories (Burlingame, CA). As described previously,5 (link),15 (link) normal mouse spleen tissue served as positive staining controls. Negative controls included omission of the primary antibody and analysis of mouse CNS tissues from unimmunized mice.
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Histological Analysis of Neuroinflammation in Mice
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- Removal of brains, spinal cords, and optic nerves from mice
- Inflammatory foci (>10 clustered inflammatory cells) in meningeal and parenchymal regions, as counted by a blinded observer (R.A. Sobel)
- Immunohistochemical staining of sections with anti-CD3 and anti-CD45R (B220) antibodies
- Fixation of tissues in 10% neutral-buffered formalin
- Paraffin-embedding and sectioning of tissues
- Use of normal mouse spleen tissue as positive staining controls
- Omission of primary antibody and analysis of mouse CNS tissues from unimmunized mice as negative controls
- Normal mouse spleen tissue
- Omission of primary antibody
- Mouse CNS tissues from unimmunized mice
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