Image acquisition and analysis

All images were acquired using an inverted Zeiss LSM 800 Airyscan confocal microscope or an upright epifluorescence Nikon Eclipse NiE microscope. Infected regions (FR positive areas of the neocortex) on the left hemisphere were identified using a 10x objective. Subsequently, all fluorescent images were taken with a 20x objective (numerical aperture = 0.5), image stacks acquired with a z-step of 2 μm and image resolution of at least 1,024 × 1,024 pixels at least 200 μm from the injection site. Images were also taken contralateral to the injection site, at homotypic locations. A minimum of three images were acquired from each section/hemisphere. For quantification purposes, the acquisition and analysis settings were kept constant for all images.

Image analysis was performed using FIJI software². All settings remained constant for each marker of interest. Each GFAP or IBA1 positive profile was co-localized with the nuclear stain DAPI to confirm its cellular nature. Total area immunostained (μm²), percentage area immunostained (total area normalized to the image size), and neuronal counts, were achieved using thresholding techniques to segment the regions of interest for analysis. To test for these microglial morphological changes, we thresholded the images so that processes were eliminated and the % area occupied by IBA1 immunoreactive cell bodies was measured. For each stack of images, the z-series was flattened to produce a maximum projected 2D image. Pre-processing was performed by adjusting the brightness and contrast, or subtracting the background, to make the fluorescent areas easily distinguishable. Areas of interest were then outlined by adjusting the threshold to create a binary image. Subsequently, various filters were applied (e.g., ‘unsharp,’ ‘despeckle,’ ‘Otsu’) before the final analysis. The masked outline of each region was created to confirm that the parameters chosen were optimal.