RNA in situ hybridization (RNAScope)

Tissue processing and embedding were performed using standard techniques. Tissues were fixed in 10% formalin overnight, paraffin embedded and cut into 5 μm sections. RNA in situ hybridization was performed with RNAscope 2.5 HD Detection Kit (RED) (Advanced Cell Diagnostics, Newark, CA) following manufacturer’s instructions and, as previously described⁷¹. The tissue sections were hybridized with probes spanning mouse Amhr2 and human and mouse Nr5a2 mRNA (Advanced Cell Diagnostics) in the HybEZ hybridization oven (Advanced Cell Diagnostics) for 2 h at 40 °C, following a series of pretreatment steps. The mouse Amhr2 probe (Advanced Cell Diagnostics catalog number 489821) targets nucleotides 914–1809. The human Nr5a2 probe (catalog number 490261) targets nucleotides 773–1786 of the human sequence. The slides were then processed for standard signal amplification steps per manufacturer’s instructions. Red chromogen development was performed following the RNAscope 2.5 HD detection protocol. The slides were then counterstained in 50% hematoxylin for 2 min, air-dried and cover slipped with EcoMount (Biocare Medical, Pacheco, CA).