Af2944

After deparaffinization and antigen retrieval, 5% bovine serum was used to block sections at RT for 1 h, and specific primary antibody SYCP1 (1:200, ab15087; Abcam), γH2AX (1:400, 05-636; Millipore), PLZF (1:100, AF2944; R&D), SOX9 (1:500, AB5535; Millipore), STRA8 (1:200, ab49405; Abcam), SYCP3 (1:200, ab15093; Abcam), PIWILI1 (1:200, A2150; Abclonal), F-ACTIN (1:200, Ab130935), DDX4 (1:200, Ab13840) was used to incubate with sections at RT or overnight at 4°C. After washing the sections three times, the slides were incubated with the corresponding secondary antibody, fluorescent dye–conjugated FITC (1:150, 115-095-003; Jackson) or TRITC (1:150, 115-025-003; Jackson) for 1 h at RT (avoiding the light). DAPI was used to stain the nucleus. All images were captured with confocal microscopy (Leica TCS SP8).

The first antibody against Sox9 for WB and IF was purchased from Invitrogen (AB5535, Millipore, Billerica, MA); anti-espin for WB and IF (611656, BD Biosciences, New York City, NY, USA); anti-β-tubulin for WB and IF (AB0039, Abways, Beijing, China); anti-E-cad for WB (3195, CST, Boston, MA, USA); anti-N-cad for WB (NBP1-48309,Novus Biologicals, Littleton, CO, USA); anti-β-catenin for WB (610153, BD Biosciences, New York City, NY, USA); anti-VIM for WB (AF2105, RD,Minneapolis, MN, USA); anti-β-actin for WB (ab8226, Abcam, Cambridge, UK); anti- GAPDH for WB (Abcam, ab9485, Cambridge, UK); anti-Ddx4 for IF (ab13840, Abcam, Cambridge, UK); anti-Plzf for IF (AF2944, RD, Minneapolis, MN, USA); anti-γH2AX for IF (ab26350, Abcam, Cambridge, UK).

Testes were directly embedded in tissue freezing medium, sectioned at 5 µm, and fixed with 4% paraformaldehyde. Slides were blocked with bovine serum (Sigma), and stained with primary antibodies. The primary antibodies used were as follows: anti-PEX16 (1∶200; PA5-60311, Invitrogen), anti-ABCD1 (1∶200; 11159-1-AP, Proteintech, Manchester, UK), anti-ABCD3 (1∶200; PA1-650, Invitrogen), anti-TEX14 (1∶500; ab41733, Abcam, Cambridge, UK), anti-DDX4 (1∶200; ab27591, Abcam), anti-γ-H2AX (1∶200; ab26350, Abcam), anti-SOX9 (1∶200; AB5535, Millipore, Billerica, MA, USA), anti-PLZF (1∶200; AF2944, R&D Systems, Minneapolis, MN, USA). Secondary antibodies were FITC- or TRITC-conjugated goat anti-mouse or anti-rabbit IgG and donkey anti-goat IgG (1∶1000; Beijing Zhongshan Biotechnology Co., Beijing, China). The slides were visualized using a ZEISS LSM800 (ZEISS, Jena, Germany) microscope.

Samples were fixed in 4% paraformaldehyde (PFA), dehydrated in graded ethanol (70-100%) and embedded in paraffin. Sections were blocked in 10% goat serum and incubated with the following primary antibodies: anti-PLZF (diluted 1:100 in TBST, AF-2944, R&D Systems, USA) anti-γH2AX (diluted 1:100 in TBST, 16-202A, Merck Millipore, USA) and anti-SOX9 (diluted 1:100 in TBST, AB5535, Merck Millipore, USA). Nuclear DNA and acrosomes were stained with 4’,6-diamidino-2-phenylindole (DAPI, F6057, Sigma–Aldrich, USA) and FITC-conjugated peanut agglutinin (PNA, RL-1072, Vector Labs, USA), respectively. Sections were analyzed using a fluorescence microscope (Leica DM300, Wetzlar, Germany).

The following antibodies were employed for immunostaining or Western blotting: rabbit polyclonal anti-FTO (Proteintech, 27226-1-AP, for Western blotting 1:3000, for immunofluorescence or immunohistochemistry 1:200), rabbit polyclonal anti-SOX9 (Millipore, AB5535, for immunofluorescence 1:400), goat polyclonal anti-PLZF (R&D, AF2944, for immunofluorescence 1:200), rat monoclonal anti-Ki67 (Invitrogen, 14-5698-95, for immunofluorescence 1:500), mouse monoclonal anti-SYCP3 (Abcam, ab97672, for immunofluorescence 1:500), rabbit polyclonal anti-STRA8 (Abcam, ab49602, for immunofluorescence 1:200), rabbit polyclonal anti-AR (Abcam, ab74272, for Western blotting 1:100), rabbit polyclonal anti-JMJD1C (ABclone, A20153, for Western blotting 1:500), and rabbit polyclonal anti-INSL3 (ABclone, A5728, for Western blotting 1:500).

We analysed the tissue expression patterns of membrane proteins identified via the BioGPS database. Several membrane genes highly or uniquely expressed in the mouse testis were chosen for validation via RT-PCR. cDNA from 11 mouse tissues (testis, heart, brain, thymus, stomach, spleen, liver, lung, kidney, ovary and uterus) was PCR-amplified with specific primers (Table1), and mouse Gapdh was used as the control.
For whole-mount staining, seminiferous tubules of mouse testis tissues were dissected at post-partum day 2.5 and 28. The prepared seminiferous tubules were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min., washed three times with PBS for 15 min. each time, and then blocked with 5% BSA for 2 hrs at room temperature. Following incubation with rabbit anti-PLD6 (ab170183; Abcam) and goat anti-PLZF (AF2944; R&D Systems) overnight at 4°C, tubules were incubated with AlexaFluor 488-labelled donkey anti-rabbit IgG and AlexaFluor 555-labelled donkey anti-goat IgG (Invitrogen) at a 1:1000 dilution for 2 hrs at room temperature. For negative controls, the primary antibody was replaced with normal rabbit and mouse IgG. The nucleus was stained with 5 μg/mL Hoechst H33342 (Sigma-Aldrich) for 30 sec. All samples were observed under a ZEISS LSM 710 microscope (Carl Zeiss).