Channel dye separation module

Histocytometry analysis was performed as previously described, with minor modifications (Gerner et al., 2012 (link)). Briefly, multiparameter confocal images were first corrected for fluorophore spillover using the built-in Leica Channel Dye Separation module. For acquisition of single stained controls, UltraComp beads (Affymetrix) were incubated with fluorescently conjugated antibodies, mounted on slides, and imaged. Cell surfaces were created using Jojo1 nuclear staining using the Imaris surface creation module, and the object statistics were exported to Excel (Microsoft). Object statistics were combined into unified CSV files and finally imported into FlowJo software for cellular gating and analysis. For correlation analyses, histocytometry data are spatially subdivided into cylindrical neighborhoods via virtual raster scanning (CytoMAP, (Stoltzfus et al., 2020 (link))). Pearson correlation coefficients are then calculated for the numbers of cells in these neighborhoods. For visual clarity, presented images were manipulated in Imaris and PowerPoint (Microsoft), with identical manipulation applied across experimental groups.

All samples were imaged using a Leica confocal SP8 microscope, with either a 40X 1.3NA (HC PL APO 40x/1.3 Oil CS2, for 20 mm sections) or a 20X 0.75NA (HC PL APO 20x/0.75 IMM CORR CS2, free working distance = 0.68 mm, for thick cleared slices) oil objective with type F immersion liquid (Leica, refractive index n_e = 1.5180). After acquisition, stitched images were compensated for spectral overlap between channels using the Leica Channel Dye Separation module in the Leica LASX software. For single stained controls, UltraComp beads (Affymetrix) were incubated with fluorescently conjugated antibodies, mounted on slides, and imaged with the same microscope settings used in the image they were being used to compensate. In all figures, for visual clarity, thresholds were applied to the displayed channel intensities.