RNA double in-situ was performed using Quantigene ViewRNA tissue ISH (Affymetrix, USA) following manufacturer’s recommended protocol. Fresh unfixed brain tissues were frozen in OCT blocks using dry-ice isopentane slurry. Brains can be stored in −80C until cryosectioning. Cryosectioning was done on Leica cryotome at 12um thickness, and sections collected on charged glass slides. Custom and off-the shelf branched-DNA oligo ISH probes were designed and synthesized by Affymetrix Quantigene ViewRNA. Sections on slides were postfixed just prior to ISH, and in-situ steps were followed according to manufacturer’s recommended protocol. For dual signal detection QuantiGene Type-1 and Type-6 probes were used. Fluorescent signals from Type-1 and Type-6 ISH probes were imaged on tile-scanning mode using Perkin Elmer spinning disk confocal at 10× magnification and auto-stitched using Volocity software. Stitched images were exported as TIFFs for further processing and adjustments to brightness and contrast in FIJI (Fiji is just ImageJ) and assembled in Adobe Illustrator.
