Metamorph advanced software

Fluorescence micrographs were obtained using an Olympus IX70 microscope equipped with a Lumen Dynamics XCite 120PC-Q fluorescence source and a Q Imaging EXi Aqua camera with an objective magnification of 20× coupled with a 1.6× magnification booster at an exposure time of 50 ms (Cy3) and 350 ms (Cy5), respectively. Images were obtained in grey scale using MetaMorph Advanced software, version 7.7.8.0 (Molecular Devices, LLC) and subsequently false-colored using ImageJ software (NIH, Bethesda, MD). Where noted, a GeneTac UC 4 × 4 microarray scanner (Genomic Solutions) was also used to visualize arrays. Acquisition settings are summarized in Supplementary Table 4, while false-color palettes used in image preparation are provided in Supplementary Figure 2.

Probe sequences of mouse Sst conjugated to Alexa Fluor 488, and Eif2a conjugated to 546 were custom-made (Advanced Cell Diagnostics, Hayward, CA). Fresh-frozen cryostat sections (10 μm) were placed on slides and fixed in cold 4% paraformaldehyde for 1 hour, followed by dehydration in an ethanol series. Tissue sections were then dried at room temperature for 30 min, followed by protease digestion at room temperature for 10 min. Using RNAscope Fluorescent Multiplex Reagent Kit (Advanced Cell Diagnostics; # 32085), tissue sections were then rinsed in deionized water, and immediately treated with target probes, preamplifier, amplifier, and label probe in a HybEZ hybridization oven (Advanced Cell Diagnostics, Hayward, CA). Images were acquired using an Olympus IX71 fluorescent microscope (Olympus, Tokyo, Japan) and a Fast 1394 CCD Camera (QImaging, BC, Canada). Overlapping signals from different fluorophores were separated and visualized under a 20x objective lens with MetaMorph Advanced software (Molecular Devices, CA, USA). The ratio of overlapping fluorescent signal areas from EIF2A and SST mRNA transcripts was determined in the cingulate cortex, based on 5 microscopic images from 3 tissue sections of each animal.