Confocal tile scan images were imported into Imaris version 9.3 (Bitplane). A surface of the peri-sinus region, including the superior sagittal and transverse sinuses, was made by manual drawing using the surface function, and the area in μm2 was determined by exporting surface statistics. A quantification of peri-sinusoidal cell numbers was generated by identifying and thresholding on positively stained cells within the three-dimensional surface of each respective channel using the spot-detection function. Quantification of GFP-C. albicans in hippocampal images was done by thresholding on and counting GFP+ puncta using the same function. Statistics were then exported into Excel (Microsoft) for further analysis.
Histocytometry plots were generated by either creating a surface of the peri-sinus region as above or the entire meningeal whole-mount, creating a value-based visual surface for all positively stained cells in each channel. This allows for quantification of fluorescent intensity and frequency of labeled and unlabeled cells, as well as their position in an x-y plane. Channel statistics were then exported into Excel (Microsoft), and position of each cell and mean voxel fluorescence, when indicated, was plotted in FlowJo software by utilizing the text to FCS conversion function (Tree Star).
Histocytometry plots were generated by either creating a surface of the peri-sinus region as above or the entire meningeal whole-mount, creating a value-based visual surface for all positively stained cells in each channel. This allows for quantification of fluorescent intensity and frequency of labeled and unlabeled cells, as well as their position in an x-y plane. Channel statistics were then exported into Excel (Microsoft), and position of each cell and mean voxel fluorescence, when indicated, was plotted in FlowJo software by utilizing the text to FCS conversion function (Tree Star).