Adl67

Transfected OSCs were plated on fibronectin-coated coverslips overnight. Cells were washed with PBS, fixed in 4% PFA (Alfa Aesar) for 15 min at room temperature and rinsed in PBS. Fixed cells were permeabilised in PBS + 0.2% Triton X-100 for 10 min, rinsed with PBS and then incubated in PBS + 0.1% Tween-20 (PBST) + 1% BSA for 30 min. Primary antibodies were diluted in PBST + 0.2% BSA and incubated overnight at 4°C. Coverslips were washed 3 × 5 min in PBST and then incubated with secondary antibodies (Invitrogen) diluted in PBST + 0.2% BSA for 1 hr at room temperature. Coverslips were washed 3 × 5 min in PBST, stained with DAPI (1:1000 diluted in PBS, Invitrogen D1306) for 10 min, washed twice in PBS and mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, P36961). The following antibodies were used: anti-HA (1:1000; abcam ab18181) anti-FLAG (1:1000; Cell Signaling Technology 14793S) and anti-Lamin (1:200; Developmental Studies Hybridoma Bank ADL67.10). Secondary antibodies (1:500) were Alexa Fluor 488-, 555- and 647-conjugated anti-mouse and anti-rabbit (Invitrogen).

Ovaries were dissected and ovarioles fixed as described above (see DAPI staining). Following fixation, ovarioles were blocked in 200 µl blocking solution for 2 h at room temperature. Ovarioles were transferred into 100 µl of PBSTx (PBS + 0.1% Triton X-100) containing mouse anti-Lamin primary antibody (1/100; ADL67.10; Developmental Studies Hybridoma Bank) and left rotating at room temperature overnight. The following day, ovarioles were washed three times in PBSTx for 10 min each and transferred into 100 µl secondary antibody solution (1/250 in PBSTx; Alexa Fluor 488 AffiniPure Donkey anti-mouse IgG; Jackson Immuno Research) including DAPI (0.4 µg/ml; Sigma). Incubation was allowed at room temperature overnight as before. The following day, ovarioles were washed (3 × 10 min in PBSTx) and mounted onto slides.