Semi-quantitative analysis of mRNA duplex staining in rat brain tissues using ImageJ

Experimental rat brain tissue sections subjected to duplex staining for INMT and AADC mRNA and positive and negative control probes on adjacent brain sections (images in Figs 2 and ^S1) were quantified using ImageJ to estimate the fraction of total area in pixels of mRNA signals by summating both colormetric channel pixels as per a prior publication analyzing this variable in brightfield images of rat FFPE brain tissues following RNAscope in situ⁵². Results of mRNA quantification are presented in Fig. ^S1 (summated INMT and AADC signals for comparison of experimental versus control tissue sections). Briefly, blue channel staining was quantified in all Figs 2 and ^S1 images with the following Color Threshold command adjustments (under the Adjust command). All default settings were used, except hue was set for 0–163, saturation was set for 0 to 90–255, depending on background, and brightness was set for 0–215 to 255, depending on background. Similarly for red channel staining, all default settings were used, except HSB color space was changed to YUV, and Y was set for 0–135 to 200, depending on background, U was set for 0–255, and V was set for 135–185 to 255, depending on background. Particles were then analyzed using the Analyze Particles command and default settings therein along with the Summarize box option checked. Outputs from the Summarize box for %Area for both colormetric channels were entered into GraphPad Prism Version 7.0a for Mac OS X to construct graphs in Fig. ^S1. All images were processed beforehand with the same microscope settings and Adobe Photoshop CC 2015.5 (Adobe Systems Inc.) filters.