A Leica DM500 microscope equipped with high-resolution digital camera (Leica MC120 HD) and MBF software was used for image acquisition. The number of Iba1-positive cells was determined in regions of interest (ROI) of 500 μm2 (2–4 sections/mice), including the cerebellar white matter (WM), internal granule layer (IGL), Purkinje cell layer (PCL) and molecular layer (ML), both for anterior (II–III) and posterior lobules (IX–X). Microglia were also classified as ramified, hypertrophic, bushy or ameboid based on their morphologies (Martini et al., 2020 (link); Savage et al., 2019 (link)): (1) Ramified—small round soma and extended, highly branched processes; (2) Hypertrophic—larger soma and thicker, shorter and less branched processes; (3) Bushy - swollen and enlarged soma, and an excess of thicker, shorter processes with few branches; (4) Stout/Ameboid - retracted processes and irregular shape.
The quantification of Iba1-positive cells of the olfactory bulb was performed by outlining 500 μm2 ROI that included the GCL. Cells and phagocytic cups were manually counted on acquired images at 20X magnification, by using the cell counter plugin from the ImageJ/Fiji NIH software (Version 1.51, National Institutes of Health).
The quantification of Iba1-positive cells of the olfactory bulb was performed by outlining 500 μm2 ROI that included the GCL. Cells and phagocytic cups were manually counted on acquired images at 20X magnification, by using the cell counter plugin from the ImageJ/Fiji NIH software (Version 1.51, National Institutes of Health).
