Quantitative Demyelination Analysis in Mice

To evaluate demyelination in the mouse brain and spinal cord, fixed tissues were stained using the Luxol Fast Blue Stain Kit (Abcam), as described in the manufacturer’s protocol. Mice were sacrificed and assessed at the peak EAE stage, approximately 17–19 days post immunization. The sections were incubated with 0.1% Luxol fast blue solution at 24℃ overnight and differentiated in 0.05% lithium carbonate solution at 24℃. When differentiation was completed, the sections were counterstained with 0.1% cresyl echt violet solution for 3 min at 24℃. Analysis of demyelination was conducted in both the brain corpus callosum region and the thoracic to lumbar region of spinal cord. The demyelinated regions in the white matter were quantified using ImageJ (NIH, USA) [37 (link), 38 (link)].