Mouse brains were extracted, flash frozen in liquid nitrogen, and embedded in OCT (Sakura Tissue-Tek ref 4583). Frozen, 14 μm, coronal sections were cut on a cryostat (Leica CM 1950) and processed for 3-color smFISH according to the ACD RNAScope multiplexed fluorescent protocol for fresh frozen tissue (ACD user manual document numbers 320513 & 320293). Briefly, sections were post-fixed in 4% PFA (Electron Microscopy Sciences) in PBS for 15 minutes, followed by alcohol dehydration. Sections were permeabilized with the proprietary protease cocktail in “pretreat IV” followed by target probe hybridization (Key Resources). For each experiment, ACD 3-plex positive control and 3-plex negative control probes were run alongside target probes to ensure tissue quality and control for background respectively. Probes were visualized with the ACD “Alt-B” color module across all experiments. For puncta counting experiments, stacks were acquired Leica SP8 at 63× magnification (n= 4–10 Z planes, 1 μm steps). In situ expression was quantified by smFISH puncta counting in Pvalb+ cells using maximum projections through confocal stacks acquired in Frontal Cortex. Soma were manually segmented based after Gaussian-filtering Pvalb puncta.
