In situ hybridization on human tissue

For each double-labeling experiment, sections from four different tissue donors were stained. To avoid bias, complete DRG sections were imaged, and individual images stitched together using NIS elements (Nikon) to generate an image of the whole DRG. Initially, fluorescent channels of the stitched images were handled separately and visually analyzed for neuronal expression of stained markers in ImageJ. A neuron, defined by a broad DAPI-positive neuronal nucleus and/or a dense array of surrounding DAPI-positive satellite glia nuclei, was manually counted as positive only when >5 puncta per cell were present. Post counting individual fluorescent channels, the channels were merged, and potential co-expression of marker genes was assessed permitting blind quantification of co-expression. Data are expressed as mean percentage and standard error of the mean calculated across the four tissue donors and as aggregated number of positive neurons from all four tissue donors.