4.5.2. RNAscope

Hemisections processed for detection of mRNA for kisspeptin, MC3R, and MC4R were analyzed using an LSM 880 laser scanning confocal microscope (Zeiss). For each section (three sections per wether), z-stacks (1.0-µm optical sections) were captured in the ARC using a Plan Achromat 20x/0.8 objective, with consistent acquisition settings used for all images. Confocal images were imported into Zen 3.0 software (Carl Zeiss Microscopy), where the total number of cells expressing kisspeptin, MC3R, or MC4R mRNA were identified by a single observer. Markers placed on cells ensured that the same cell was not counted twice, and cells in which complete cell bodies were visible were included in the analysis, with DAPI used to visualize nuclear area. Images containing marked cells were imported in to FIJI/ImageJ software, where the numbers of cells were quantified. The degree of double or triple labeling was calculated as a percentage of the total number of kisspeptin cells expressing MC3R and/or MC4R, the total number of MC3R cells expressing kisspeptin and/or MC4R mRNA, and the total number of MC4R-expressing cells containing kisspeptin and/or MC3R mRNA.