*4.5. In Situ Hybridization*

For in situ hybridization, the probes were labeled by Roche DIG RNA labeling kit (Roche, #11175025910, Germany) following instructions of the manufacturer. Ovaries were dissected in PBS, and then fixed in 4% PFA overnight. After washing 3 times with PBT (PBS + 0.1% Tween 20), the tissues were again washed with methanol/PBT for 5 min and rinsed three times with PBT. After the samples were rinsed with 1:1 hybridization buffer/PBT for 5 min, 100% hybridization buffer for 5 min, and three times with PBT for 5 min each, respectively, the DIG-labeled RNA probes were pre-hybridized at 100 ◦C for 1 h prior to hybridization. For the hybridization, the tissues were incubated overnight with a probe at 60 ◦C. After hybridization, to wash off the unspecific binding, the tissues were rinsed with washing buffer four times for 30 min, and then washed with MABT buffer two times for 10 min. After blocking with 5% blocking solution, the tissues were incubated with anti-DIG-POD (1:200; Roche) in PBT (with 0.5% blocking solution) overnight. After washing with MABT for 1 h, we added 1 μL diluted fluo-dye (Roche) in amplification buffer into the tissue solution and kept it at room temperature for 1.5 h. Following the procedures of in situ hybridization, the immunostaining was carried out as previously mentioned [28]. Observations were carried out with an upright confocal microscopy.
