Anti fade fluorescence mounting medium

Briefly, ovaries were digested with 1% trypsin and dispersed ovarian cells were collected in 1% PFA containing 2% Triton X-100, then dripped on a slide and fixed for 6 h at room temperature. The slides were blocked with ADB (antibody dilution buffer; containing 1% normal donkey serum, 3‰ BSA, and 1‰ Triton X-100 in TBS) for 30 min at room temperature and incubated with the appropriate primary antibodies overnight at 37 °C. Primary antibody dilution: SYCP3 (1:50; #sc-20845, Santa Cruz Biotechnology, CA, USA), RAD51 (1:50; #sc-8349, Santa Cruz Biotechnology, CA, USA), and γ-H2AX (1:100; #NB100-2280, NOVUS, CO, USA) (Additional file 7: Table S3). The slides were rinsed thoroughly in PBS and incubated with second antibodies (1:200; Life Technologies, USA) dissolved in PBS for 1 h at 37 °C. The sections were then rinsed in PBS, stained with Hoechst 33342 (B2261, Sigma, USA) for 5 min, and sealed in anti-fade fluorescence mounting medium (Applygen, China) with coverslips. The sections were examined and photographed using Nikon Eclipse 80i digital fluorescence microscope. The average percentages of germ cells staining with designated antibody were determined from three slides, each of which included at least three ovaries. Germ cells were randomly dispersed on the slides, and at least 300 germ cells were analyzed per slide. Unidentified germ cells were not included in the analysis.

Collected ovaries were fixed overnight at 4°C in 4% paraformaldehyde for immunohistochemistry. Samples were treated through an ethanol series and xylene, infiltrated with paraffin wax, and sliced into 5 μm thick sections. Sections were dewaxed in xylene and rehydrated in ethanol series, and then were dyed with hematoxylin for 1 min. For immunofluorescence staining, sections were heated on high power for 4 min once and on low power for 4 min three times in 0.01 M sodium citrate (pH 6.0) for antigen retrieval. Then sections were blocked for 1 h in Immunol Staining Blocking Buffer (P0102) at room temperature. Primary antibody were diluted in PBS and sections were incubated overnight at 4°C (antibody information is listed in Table S1). Then samples were incubated with secondary antibodies, either Alexa Fluor 488-conjugated, 555-conjugated anti-mouse, anti-goat, anti-rabbit or anti-chicken (1:100; Invitrogen), for 80 min at 37°C. Hoechst 33342 (B2261; Sigma) or PI (Propidium Iodide Solution, 421301, BioLegend) were used for nuclear DNA. Sections were coverslipped with anti-fade fluorescence mounting medium (Applygen, Beijing, China). The images were acquired on a confocal microscope (Eclipse, Nikon, Japan) and analyzed by the NIS-Elements BR 3.2 software.

Ovaries were fixed in 4% PFA overnight, embedded in paraffin, and sectioned at 5 μm. After dewaxing, rehydration, and high‐temperature (92°C) antigen retrieval with 0.01% sodium citrate buffer (pH 6.0), the sections were blocked with 10% normal serum for 60 min at room temperature and immunostained with primary antibodies overnight at 4°C. Subsequently, after rinsing thoroughly with PBS, the slides were then incubated with Alexa Fluor 488‐ or 555‐conjugated secondary antibodies (1:100; Invitrogen) and Hoechst 33342 (1:1000; B2261, Sigma) at 37°C for 1 hr. Slides were then rinsed in PBS and sealed in antifade fluorescence mounting medium (Applygen) with coverslips. Sections were examined and photographed using a Nikon Eclipse 80i digital fluorescence microscope or a Nikon A1 laser scanning confocal microscope. Primary antibodies and dilutions used are presented in Table S2.