RNAscope for Specific Gene Expression

Brains were quickly isolated and fixed in 10% neutral‐buffered formalin (NBF) for 24-h, followed by ethanol gradient dehydration and infiltration with melted paraffin in an automated processor. An array of 4 µm coronal sections of whole-brain was prepared, to collect different regions of the cortex, and RNAscope in situ hybridization was performed according to the manufacturer’s reference guide, using the single‐plex RNAscope® assay kit-BROWN (Advanced Cell Diagnostics) on a Leica Biosystems BOND RX platform, as described previously^{64 (link)}. Brain slices were baked and deparaffinized on the instrument, followed by target retrieval and protease treatment. Hybridization was performed by using probe Mt1 (ACD, 547711), Tsc22d3 (ACD, 448341), Gjb6 (ACD, 458811) and Atp1b2 (ACD, 417131) followed by amplification as these genes were among our top hits (based on FDR and fold changes), DAB chromogenic detection and counterstain with haematoxylin. Imaging of sections was carried out on a Leica Biosystems Aperio AT2 Digital Pathology Scanner and analysis performed using Visiopharm software.