Ab209710

The sequence-specific DNA fragments of lnc196, lnc304, and lnc172 were amplified with a pair of primers whose reverse primer contained the core sequence of T7 promoter at the 5′ end (primer and lncRNA sequence information in Table S1). Digoxin-labelled antisense RNA probes were synthesized using the DIG-RNA labeling kit (Roche, Mannheim, Germany). The gonads were fixed in 4% PFA at room temperature for 2 h, washed in PBS for 3 × 10 min, and embedded with frozen section compound (Sakura Finetek, Torrance, USA). The samples were sectioned with a thickness of 10 μm. The slides were dried at 37 °C for 30 min, rehydrated, and washed in PBS three times and hybridized with DIG-labeled anti-sense probes at 60 °C for 16 h. After a series of washing steps, the slides were blocked in 2% blocking reagent (Roche, Mannheim, Germany) in MAB buffer (0.1 M Maleic acid and 0.15 M NaCl, pH 7.4) for over 2 h at room temperature and incubated in anti-DIG-POD FAB fragments (Roche, 1:2000 diluted in 1% blocking solution) and anti-vasa antibodies (abcam, ab209710, 1:400) at 4 °C overnight. After washing in PBT and PBS buffer three times, the slides were incubated in TSA™ Plus Fluorescence Systems (Red) (PerkinElmer), FITC-conjugated goat anti-rabbit IgG (H + L), and DAPI (1 μg/mL) for 1 h. The images were acquired with a Leica Stellaris 5 confocal microscope (Leica, Mannheim, Germany).

The localization of Vasa in gonads of both the triploids and diploids at 360 dph was detected using Vasa antibody (ab209710, Abcam, UK). Specifically, the gonadal samples fixed in Bouin’s solution were embedded in paraffin wax, and then sliced into 5–7 um serial sections. The tissue sections were rehydrated with PBS, digested with proteinase K (10 ug/mL) for 30 min, and then fixed in 4% PFA for 20 min, washed with PBST for 5 min four times. Subsequently the samples were incubated with Vasa antibody at a 1: 1000 dilution at 4 ℃ overnight, then washed in PBST and incubated with the secondary antibody at a 1:2000 dilution for 30 min at room temperature. Finally, the samples were stained with DAPI (E607303, Sangon, China) for 10 min, and washed in PBST. Fluorescence images were captured with a fluorescence microscope (model BX-51N-34FL, Olympus, Japan).