Ab34139

Formaldehyde-fixed tissue sections, DDX4^C25-positive cells and oocytes were incubated with antibodies for the detection of DAZL (ab34139; Abcam), DDX4 (DDX4^C25 antibody, ab13840; Abcam and DDX4³⁵¹ antibody, 17545-1-AP; ProteinTech, UK), DPPA3 (ab19878; Abcam), IFITM3 (ab15592; Abcam) and PRDM1 (PA5-20310; ThermoFisher, UK). All images were acquired using a Leica SP8 microscope with hybrid detectors and x63 oil immersion lens. Fluorochromes were imaged sequentially. Images were then analysed on ImageJ (NIH, USA).

Testis tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 5 µm sections. The paraffin sections were heated in an autoclave in citrate buffer and preincubated in permeabilization blocking buffer (0.1 mmol/L PBS, pH 7.3, 0.5% (wt/vol) Triton X-100). The sections were blocked for 60 min with 10% (vol/vol) donkey serum. The testicular sections were stained with primary antibodies at 4 °C overnight and secondary antibodies for one hour at room temperature, followed by washing and staining with DAPI (Beyotime, Jiangsu, China) for 10 min at room temperature; then, the sections were observed under a confocal microscope (Zeiss LSM710, Carl Zeiss Microscopy GmbH, Jena, Germany) at excitation wavelengths of 488 nm (green) and 405 nm (blue). The primary antibodies were as follows: rabbit polyclonal anti-DAZL (1:100; ab34139, Abcam, Cambridge, UK); mouse monoclonal anti-SYCP3 (1:100; ab97672, Abcam); rabbit polyclonal anti-TNP1 (1:300; ab73135, Abcam); rabbit polyclonal anti-PGK2 (1:300; D121803, Sangon Biotechnology, Shanghai, China) and rabbit monoclonal anti-WT1 (1:50; ab89901, Abcam). The secondary antibodies were as follows: Cy3-conjugated AffiniPure donkey polyclonal anti-mouse IgG (H + L) and Alexa Fluor 488-conjugated AffiniPure donkey polyclonal anti-rabbit IgG (H + L) (both 1:500; Jackson ImmunoResearch Laboratories, Philadelphia, PA, USA).

Immunofluorescent staining of embryonic sections was carried out as described previously [50 (link)]. Briefly, whole embryos or cultured urogenital organs were fixed at 4°C overnight in 4% paraformaldehyde, paraffin embedded, and sectioned. Slides were then dewaxed, rehydrated, and antigen-retrieved by microwaving in citrate buffer (10mM sodium citrate, 0.05% Tween 20, pH6.0). After blocking, slides were incubated with primary antibodies at 4°C overnight. Slides were then incubated with donkey secondary antibodies conjugated to FITC, Rhodamine Red X or DyLight 649 (Jackson ImmunoResearch) and mounted with ProLong Gold Antifade reagent with DAPI (Life Technologies).
Primary antibodies against GATA4 (sc-25310, Santa Cruz Biotechnology), DAZL (ab34139, Abcam), SSEA1 (MAB4301, Millpore), MVH (AF2030, R&D Systems), GCNA (a gift from George Enders, University of Kansas Medical Center, Kansas City, KS) [51 (link)], SOX2 (ab97959, Abcam), NANOG (IHC-00205, Bethyl Laboratories), OCT4 (560186, BD), MILI (a gift from Gregory J. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) [52 (link)], and SYCP3 (sc-33195, Santa Cruz Biotechnology) were used in the study.

Protein samples were extracted from testes and the cultured cells using RIPA buffer and following the standard protocol. After quantification, proteins were separated by SDS-PAGE, and Western blot analysis was performed as previously described [23 (link)]. The primary antibodies used were mouse anti-UCHL1 (1: 1,000; ab8189, Abcam), rabbit anti-PLZF (1: 1,000; ab104854, Abcam), mouse anti-THY1 (1: 1,000; ab205719, Abcam), rabbit anti-VASA (1: 1,000; ab13840, Abcam), rabbit anti-DAZL (1: 500; ab34139, Abcam), goat anti-CXCR4 (1: 500; ab1670, Abcam), rabbit anti-SV40 (1: 1,000; 15,729, Cell Signaling Technology), mouse anti-PCNA (1: 1,000; sc-56, Santa Cruz Biotechnology), mouse anti-β-actin (1: 3,000; CW0096, CWBIO), rabbit anti-KIT (1: 1,000; 3074, Cell Signaling Technology) and rabbit anti-STRA8 (1: 1,000; ab49602, Abcam). The secondary antibodies used were goat anti-rabbit IgG-HRP (1: 2,000; CW0156, CWBIO), goat anti-mouse IgG-HRP (1: 3,000; CW0110, CWBIO) and rabbit anti-goat IgG-HRP (1: 3,000; CW0109, CWBIO). Finally, the blots were visualized with a Western Bright ECL Kit (Comwin) and chemiluminescence (Chemi-Doc XRS, Bio-Rad).

The following antibodies were used for immunoblot (IB) and immunofluorescence (IF) studies: rabbit anti-PLZF (IF, 1:1000, Abcam: ab189849), rabbit anti-DAZL (IF, 1:1000, ab34139), rabbit anti-HA (IB, 1:1000, Abcam: ab9110), mouse anti-HA 12CA5 monoclonal Antibody (IF, 1:100, Roche: AB_514505), rabbit anti-H3S10P (IF, 1:2000, Abcam: ab5176), rabbit anti-SYCP1 (IF, 1:1000, Abcam ab15090), rabbit anti-DMC1 (IF, 1:500, Santa Cruz: SC-22768), mouse anti-MLH1 (IF, 1:500, BD Biosciences: 551092), rabbit anti-Actin (IB, 1:1000, Sigma A2066), rabbit anti-GFP (IF, 1:1000, ab6556), rat anti-TRA98 (IF, 1:1000, ab82527), rabbit anti-p107 (IB, 1:1000, Santa Cruz: SC-318), rabbit anti-FOXO3 (IF, 1:200, CST 2497), rat anti-NANOG (IF, 1:1000 Thermo: eBioMLC-51), rat anti-SYCP3 and guinea pig anti-SYCP3, rabbit and rat anti-STRA8, rabbit and gunia pig anti-MEIOSIN N-terminal (a.a. 1-224) and rabbit and gunia pig anti-MEIOSIN C-terminal (a.a. 405-589) as described previously^{9 (link)}.

Western blotting (WB) was performed using a standard protocol. Whole-embryonic gonads or pieces of adult testis were lysed, and the proteins were separated in SDS–polyacrylamide gels and transferred to nitrocellulose or polyvinylidene fluoride membranes. To detect FLAG, DAZL, MVH and β-TUBULIN, the respective anti-FLAG M2 (1:10,000, Sigma-Aldrich, F3156), anti-DAZL (1:1,000, Abcam, ab34139), anti-MVH (1:1,000, Abcam, ab13840) or anti-β-TUBULIN (1:5,000, Sigma-Aldrich, 1A6) antibodies were reacted with membranes for 1 h at room temperature. After washing the membranes, the secondary antibodies, horse/goat anti-mouse/rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (1:2,000, Cell Signaling, #7076S and #7074) was reacted with membranes for 30 min at room temperature. Signals were detected using SuperSignal West Femto Chemiluminescent Substrate Kits (Thermo Scientific). Images were acquired using the Ez-Capture MG chemiluminescence imaging system (Atto, Tokyo, Japan). All uncropped WB can be found as in the Supplementary Fig. 10. The FLAG-DAZL protein signals were quantified using ImageJ software.