In situ hybridization

Sections of paraffin-embedded ovaries were deparaffinized using xylene, rehydrated twice with 100% ethanol for 5 min, and air-dried at 60°C in a dry oven for 5 min. Then, slides were processed as indicated by the manufacturer for the RNAscope Multiplex Fluorescent Assay (Advanced Cell Diagnostics, 320850). Briefly, slides were treated with hydrogen peroxide for 10 min at room temperature. Then, slides were rinsed in Milli-Q water and heated in a beaker at 98–100°C for 15 min in 1× target retrieval solution. After rinsing in Milli-Q water for 15 s, slides were transferred in 100% ethanol for 3 min. A hydrophobic barrier was created around the sections using an Immedge pen and slides were incubated with protease solution for 15 min, rinsed in Milli-Q water three to five times, and incubated with Myo10 probe (ACD, 840661) or negative control probe against Bacillus subtilis DapB (ACD, 310043) at 40°C for 2 h in a HybEZ oven. Subsequent amplification and detection steps were performed as recommended by the manufacturer and slides were taken for image acquisition using a Zeiss LSM 880 confocal microscope.