For the chromogenic in situ hybridization, brain sections containing the PVT of 14-week-old males were prepared using a mouse brain matrix with 1 mm section dividers (CellPoint Scientific, Gaithersburg, MD, USA), fixed for 28 hours in 10% formalin and embedded in paraffin. Five μm sections were cut with a microtome and single in situ hybridization (ISH) for Gck (cat. number: 400971) or double in situ hybridization for Slc17a6 (Vglut2) and Gad67 (cat. number: 319171-C1 and 400951-C1) was performed using RNAscope® probes and RNAscope® 2-plex detection kit (ACDBio, Newark, CA, USA). Sections were counterstained with Mayer’s hematoxylin and mounted using aquatex mounting medium (cat. number: 363123S, VWR, Radnor, PA, USA) and observed using an Axio Imager D1 (Zeiss, Oberkochen, Germany).
For fluorescent in situ hybridization, 14-week-old males were transcardially perfused with 4% PFA and 20 μm cryosections were prepared. Gck mRNAs were detected using RNAscope probes (Cat# 400971-C1) and RNAscope® Fluorescent Multiplex Detection Reagents following manufacturer’s instructions. Sections were mounted using DAPI-fluoromount (SouthernBiotech®, Birmingham, AL, USA) solution. Images were acquired on a ZEISS Axio Imager.M2 microscope, equipped with ApoTome.2 and a Camera Axiocam 702 mono using the AxioVision software (Zeiss, Oberkochen, Germany).
For fluorescent in situ hybridization, 14-week-old males were transcardially perfused with 4% PFA and 20 μm cryosections were prepared. Gck mRNAs were detected using RNAscope probes (Cat# 400971-C1) and RNAscope® Fluorescent Multiplex Detection Reagents following manufacturer’s instructions. Sections were mounted using DAPI-fluoromount (SouthernBiotech®, Birmingham, AL, USA) solution. Images were acquired on a ZEISS Axio Imager.M2 microscope, equipped with ApoTome.2 and a Camera Axiocam 702 mono using the AxioVision software (Zeiss, Oberkochen, Germany).