In situ hybridization (ISH)

Tissue fixation by perfusion was performed as described under IHC. Advanced Cell Diagnostics (ACD) RNAscope Technology was used for ISH. Brains were frozen in OCT and tissue was cut at 14 μm, mounted onto Superfrost Plus slides and kept at −80°C for less than three months. Tissue sections was thawed at room temperature briefly, washed with 1× PBS and subsequently processed per the ACD’s protocols. Tissue sections were then permeabilized with solutions from the pretreatment kit and incubated with protease for 30 min and hybridization probes for another 2 h at 40°C.