F9006

For immunolocalisation of PMEs and HGs with different DMs, the roots were treated with Foc1 and Foc4 for 6, 12, 24 and 48 h and harvested at the same time points to minimise the effect of other factors. The protocol for fixation, embedding of samples and immunolabelling was carried out as described by Xu et al.⁵⁶ . We labelled the sections with primary monoclonal antibodies PME, 2F4, LM19, LM20, JIM5 and JIM7. The anti-mouse lgG-FITC (F9006, Sigma) was used as secondary antibody for PME and 2F4 antibodies, and the anti-rat lgG-FITC (F6258, Sigma) was used for the other antibodies. Sections marked only with secondary antibodies were used as controls. At least three replicates were prepared for each antibody. Images were collected using a LSM 7 DUO laser scanning confocal microscope (ZEISS, Germany) and Olympus BH-2-FRCA microscope, and fluorescence signals were examined with LSM 7 DUO laser scanning confocal microscope (ZEISS, Germany) by the function “Histo” to determine an average fluorescence signal of a whole root section.

Ovarian tissues were incubated in Hypo extraction buffer (HEB; 0.6 M Tris, 0.5 M Sucrose, 0.17 M trisodium citrate dehydrate, 0.5 M EDTA, 0.5 M DTT, 0.1 M PMSF) for 1.5 h at room temperature. Tissues were then mechanically dispersed and cells transferred onto a slide fixed with 1% PFA overnight. Blocking was performed by dipping the slides in antibody dilution buffer (ADB; 0.3% BSA, 10% normal goat serum and 0.005% Triton-X-100 in TBS) for 30 min before incubation in primary antibody, anti-SCP3 at a dilution of 1:150 (Abcam, ab97672, San Francisco, CA, USA; or Novus, NB300-232, Littleton, CO, USA), anti- γH2AX at a dilution of 1:150 (Abcam, ab26350), anti-RAD51 at a dilution of 1:150 (Abcam, ab133534), BRCA1 at a dilution of 1:100 (Boster, PB9015, Wuhan, China), or anti-MLH1 at a dilution of 1:150 (BD Pharmingen, 551091, Franklin Lakes, NJ, USA) for 3 h at 37 °C. The slides were further fixed with ADB overnight at 4 °C, washed three times with TBS, before incubation in secondary antibody (Beyotime, A0521 or A0516, Nantong, China; or Sigma, F0382 or F9006) at a dilution of 1:150 dilution in ADB for 3 h at 37 °C. Hoechst 33342 was used to stain nuclei and the slides were mounted with Vectashield (Vector, H-1000, Shanghai, China).^{41 (link)} Images were taken under a fluorescence microscope (BX51; Olympus, Tokyo, Japan).