X-gal staining

The X-gal staining was performed as described previously (23). Tissues were fixed with a mixture of 2% paraformaldehyde (PFA) and 0.2% glutaraldehyde, immersed in the fixative overnight, and transferred to 20% sucrose in 20 mM phosphate-buffered saline (PBS; pH 7.4) until they sank. The tissues were frozen in liquid nitrogen and embedded in Tissue-Tek OCT compound (Sakura Finetek Japan, Tokyo, Japan), and 16-μm-thick sections were cut on a cryostat (Leica CM1850 UV; Leica, Wetzlar, Germany; HM525 NX; Thermo Fisher Scientific). Sections were washed for 10 min in PBS and incubated with X-gal staining solution {5 mM K₄[Fe(CN)₆], 5 mM K₃[Fe(CN)₆], 20 mM Tris-HCl (pH 7.3), 2 mM MgCl₂, 0.2% NP-40, 0.1% sodium deoxycholate, and 0.1% X-gal in PBS} at 37°C for 3 days. We used tissues from littermate Dst^Gt-inv/+ mice as negative controls.