Cellsense 1

Four sections that were 50 μm apart from each other were counted per mouse and three mice per genotype were quantified. The sections were imaged at 20X magnification with a slide scanner (Olympus). The entire euriochrome cyanine-stained area on every slide was outlined using cellSense 1.18 (Olympus). Demyelination was measured as demyelinated area per mouse/whole area per mouse.

To quantify the colocalization of NP with macropinosomes, cells were plated in the same way as described in the measurement of the macropinocytic index. Then, cells were incubated with 1 mg/mL TMR-dextran and 1 mg/mL Cy7 labeled NPs simultaneously. A group of cells were treated with 25 µM EIPA to evaluate the effect of macropinocytic inhibition on colocalization. After incubation, cells were fixed and sealed onto glass slides. Cell images were captured using an Olympus IX-83 inverted fluorescence microscope with a 100× phase objective. A z-stack of frames throughout the entire height of cell monolayers was acquired. The z-stack images were then collapsed to a single image using extended focus imaging projection. Background was subtracted with a constant of 1500. For each channel, the contrast was adjusted to the same scale. The Pearson correlation coefficient (PCC) between pixels of TMR-dextran and pixels of Cy7 NPs was analyzed by Cellsense 1.16 (Olympus).