In situ hybridization on whole mount sample and sections were performed as described 27 (link), 32 (link) with some minor modifications. Adult gonads were dissected and cut into small pieces, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) (4% PFA-PBS) at 4°C overnight, followed by buffer changes with 0.5 M sucrose in PBS at 4°C overnight. The samples were embedded in Optimal Cutting Temperature and cryosectioned at 8 μm for ovaries and 5 μm for testes on the Leica RM2135 Microtomes (Leica, Germany). The cryosections were mounted on frost glass slides (Fishery, USA) and stored at -80°C before use. pLcvasa854 containing the 854-bp Lcvasa cDNA fragment was linearized with Apa I and Sac II for the synthesis of sense and anti-sense riboprobes from Sp6 or T7 promoter by using the digoxigenin (DIG) or FITC RNA Labeling Kit (Roche). The probes were treated with RNase-free TURBO DNase (Ambion) and purified. Chromogenic and fluorescent in situ hybridization (FISH) was carried as described 32 (link). After extensive washes in PBS in darkness, the slides were stained for nuclei with DAPI (1 µg/ml) for 10 min at room temperature, washed three times in PBS and mounted for microscopy.