Figure 1.

Multiplex detection of RNA expression in the arcuate nucleus of control and prenatal androgen-treated mice. A, Confocal images of 12 RNA targets visualized simultaneously in representative sections from the arcuate nucleus (ARC) of PNV male, PNV female, and PNA female mice using RNAscope HiPlex fluorescent in situ hybridization. Scale bar = 100 µm. B, Representative confocal section of the ARC from a PNV female mouse containing labeling for Dapi and RNA transcripts for KISS1, TAC2, PDYN, FOS, ESR1, PGR, AR, AMRH2, TAC3R, OPRK1, SLC17A6 (vGluT2), and SLC32A1 (vGaT). Scale bar = 100 μm. (i) High magnification images of insets containing representative cells depicting automated detection of Dapi borders (ii) and RNA transcripts (iii) by Cellprofiler software. Signal that did not meet the criteria for RNA transcripts are represented by purple outlines. (iv) Dapi cells (randomly pseudocolored by CellProfiler software) containing at least 3 RNA puncta (Scale bar in inset = 10μm).