RNAscope in situ hybridization and mRNA quantification

RNAscope analysis was performed on WT and MC3R-flox mice, all injected with AVV2-hsyn-mcherry-cre. The animals were perfused as described above, however using 4% paraformaldehyde instead of 10% formalin according to ACD RNAscope recommended protocols, and 20μm sections were directly mounted onto Superfrost glass slides for RNAscope in situ hybridization. RNAscope multiplex fluorescent in situ hybridization version 2 was used according to the protocol described in the kit. MC3R mRNA expression was visualized using the probe Mm-Mc3r-C2 probe (Ref: 412541-C2). Images were obtained via confocal microscopy (Z-stack and tile scan of whole hypothalamus). The mRNA count (Figures 1C and ​and1D)1D) was performed using Fiji/ImageJ software, where a region of interest (ROI) was created and placed on the area with mRNA expression in the MH region. The Mc3r mRNA was manually counted, and the same ROI size was used for all the images.

For MC4R, LEPR, VGAT, VGlut2, AgRP, and POMC the probes utilized were: Mm-Mc4r-C3 (Ref: 319181-C3), Mm-Lepr-C3 (Ref: 402731-C3), Mm-Slc32a1 (Ref: 319191-C1), Mm-Slc17a6 (Ref: 319171-C1), Mm-Agrp-C2 (Ref: 400711-C2), and Mm-Pomc-C3 (Ref: 314081-C3) respectively. The confocal images were taken using LSM700 microscope and Zen software (z-stack and 20x zoom) and the cells were counted in the entire DMH, VMH, or ARC regions (Figures 2 and S3) using Fiji/ImageJ software. Duplicate or triplicates sections for the probes combinations were analyzed for each mouse, and the average of those measurements used to establish the number of cells which expressed the mRNA for statistical analysis. The colocalization cell count was performed considering the soma (stained with DAPI) containing at least one transcript of each probe as a positive cell. For the mRNA pixels measurement, using Fiji/Image J, a ROI was created covering entirely DMH, VMH, or ARC sections in duplicates or triplicates for each animal, and the signal was measured using the “measure” software feature. The average of the signal value for each animal was used to determine its signal intensity and used for statistical analysis.