In Situ Hybridization on Cryosections

ISH was performed on 30‐μm‐thin cryosections as described previously (Sabbagh et al., 2018 (link)). Sections were first allowed to air dry for 1hr at room temperature and washed with PBS for 5 min to remove freezing media. They were then fixed in 4% PFA for 10 min, washed with PBS for 15 min, incubated in proteinase K solution for 10 min, washed with PBS for 5 min, incubated in 4% PFA for 5 min, washed with PBS for 15 min, incubated in acetylation solution for 10 min, washed with PBS for 10 min, incubated in PBS‐diluted 0.1% triton for 30 min, washed with PBS for 40 min, incubated in 0.3% H₂O₂ for 30 min, washed with PBS for 10 min, pre‐hybridized with hybridization solution for 1 hr, before being hybridized with heat‐denatured riboprobes at 62.5°C overnight. Sections were then washed for five times in 0.2X SSC buffer at 65°C. Slides were then washed with TBS, blocked, and incubated with horseradish peroxidase‐conjugated anti‐DIG (#11207733910, Roche, RRID:AB_514500) or anti‐fluorescein antibodies (#11426346910, Roche, RRID:AB_840257) overnight at 4°C. Lastly, bound riboprobes were detected by a tyramide signal amplification system (#NEL753001KT, PerkinElmer).