Cells on Matrigel (BD Bioscience) coated cover slides were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) at 4 °C overnight, and immunofluorescence (IF) assays were performed according to published protocols55 (link). The following antibodies were used: PLZF (SC-28319, Santa Cruz Biotech, Dallas, TX, USA), STRA8 (ab49602, Abcam, Cambridge, MA, USA), SYCP3 (ab15093, Abcam), Alexa Fluor 488- or TRITC-conjugated anti-mouse and anti-rabbit secondary antibodies (115-545-146, 115-025-146; 111-545-144, and 111-025-144, Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 0.5 µg/mL DAPI before being visualized using an Olympus microscope BX53. Images were processed with the Image-J software. For histology studies, testes were fixed in Bouin’s fixative at 4 °C overnight for staining with hematoxylin and eosin, as previously described56 (link). For immunohistofluorescence (IHF), testes were fixed with 4% paraformaldehyde in PBS at 4 °C overnight and embedded in paraffin. Testis sections were stained with an ACR antibody (HPA048687, Atlas Antibodies, Sweden), followed by staining with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch) and Rhodamine-labeled Peanut Agglutinin/PNA (RL-1072, Vector Laboratories, USA). Images were collected using a fluorescent microscope (Leica, DM400BLED368424).
Sycp3
Manufactured by Abcam
Sourced in United States
SYCP3 is a protein that plays a key role in the process of meiotic recombination, which is essential for the proper segregation of chromosomes during cell division. It functions as a component of the synaptonemal complex, a structure that forms between homologous chromosomes during meiosis.
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Lab products found in correlation
γ h2ax, by Abcam (4 mentions)
Photo flo, by Kodak (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab15093, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Sangon (1 mentions)
Plzf sc 28319, by Santa Cruz Biotechnology (1 mentions)
Stra8, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Alexa fluor 488 conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Dm400bled368424, by Leica camera (1 mentions)
γh2ax, by Merck Group (1 mentions)
Linolenic acid, by Merck Group (1 mentions)
Phenol red free dmem, by Merck Group (1 mentions)
Quantitative rt pcr primers and probes, by Merck Group (1 mentions)
Plastic embedding medium, by Electron Microscopy Sciences (1 mentions)
Taq dna polymerase, by Qiagen (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ldn193189, by Merck Group (1 mentions)
11 protocols using sycp3
1
Immunofluorescence Staining of Germ Cells
2
Production and Characterization of SCML2 Antibodies
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The 6xHis-SCML2 (mouse, 147–321 aa) fusion protein was expressed in E. coli using the pQE-30 expression vector, purified with Ni-NTA resin, and used to immunize rabbits and guinea pigs at Cocalico Biologicals Inc., resulting in polyclonal antisera UP2323 (rabbit) and gp92 (guinea pig). Affinity purified SCML2 antibodies (1:1000) were used for immunofluorescence and Western blotting analyses. Other antibodies used were γH2AX (1:1000 or 1:200, Millipore), SYCP3 (1:200, Abcam), USP7 (1:200, Bethyl), and ACTB (1:7500, Sigma-Aldrich).
3
Immunofluorescence Staining of Stem Cells
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Antibodies to BMP2, SYCP3, MVH, γHis2A.X and MCM2 were obtained from Abcam (Cambridge, MA). Alexa-conjugated secondary antibodies were obtained from Invitrogen (Carlsbad, CA). Plastic embedding medium was obtained from Electron Microscopy Sciences (Hatfield, Pa). Tissue-Tek Optimal Cutting Temperature (OCT) compound was obtained from Sacura Finetek USA Inc. (Torrance, CA). Phenol red-free DMEM, linolenic acid, BSA, and fine chemicals were purchased from Sigma Chemical Company (St. Louis, MO). LDN 193,189 was purchased from Sigma Chemical Co. (St. Louis, MO). DMH1 was purchased from Tocris Bioscience (Bristol, UK). Human holo-transferrin was obtained from BD pharmaceuticals (San Jose, CA). Quantitative RT-PCR primers and probes were synthesized by Sigma Chemical Company. RNeasy mini kit and Taq DNA polymerase were from Qiagen, Inc. (Valencia, CA). The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) kit was from Roche pharmaceuticals (Mannheim, Germany). All other molecular biology grade chemicals were obtained from Sigma Chemical Company or Fisher Scientific (Pittsburgh, PA).
4
Immunofluorescence Staining of SYCP3
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Cells were fixed in 4% paraformaldehyde for 30 minutes and washed three times with PBS. The cells were treated in 0.2% Triton‐X for 10 minutes. Non‐specific adhesion sites were blocked with 3% bovine serum albumin (BSA; Sigma, Poole, Dorset, UK) for 30 minutes at room temperature. The primary and secondary antibodies were diluted in a solution of PBS containing 3% BSA, 1% horse serum and 0.1% Triton X‐100. Cells were incubated with primary antibodies SYCP3 (Abcam) overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. Nuclei were stained with DAPI (Thermo Fisher Scientific). Stained samples were then visualized, and images were captured using a LSM710 confocal microscope (Zeiss) and analysed by the Image J software.
5
Meiotic Progression Analysis in Human SSCs
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Meiotic chromatin spread assays were performed to determine the meiotic progression in human SSCs by 3D-I system according to the method described previously [24 (link)]. Cells were lysed by a hypotonic solution and spread evenly over slides layered with 1% PFA and 0.15% Triton X-100. Slides were dried for 24 h at room temperature in a humid chamber. Cells were treated with 0.04% photoflo for 5 min and blocked with 4% goat serum. Triple or duplicate immunostaining was performed in these cells using primary antibodies, including SYCP3 (Abcam, 1:100), CREST (Immunovision, 1:100), and MLH1 (Abcam, 1: 50), γH2AX (Abcam, 1:100), at 37 °C for 2 h in a humid chamber. Goat anti-rabbit Alexa Fluor 594 (Invitrogen) and goat anti-human Alexa Fluor 488 secondary antibodies (Invitrogen) were applied at 1:200 dilution and incubated for 90 min at 37 °C. Cells were washed three times with PBS, and images were captured with a fluorescence microscope (Leica).
6
Immunofluorescence Staining of Spermatocytes
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Spreads of spermatocytes and immunofluorescence staining were prepared according to the previous references51 (link)52 (link). Briefly, seminiferous tubules were incubated in hypotonic extraction buffer (50 mM Sucrose, 17 mM Sodium citrate, 30 mM Tris (pH 8.2), 2.5 mM DTT, 1 mM PMSF (pH 8.3) and 5 mM EDTA on ice for 20 minutes, minced in 100 mM sucrose, spread on slides and fixed in 1% PFA with 0.1% Triton X-100. Slides were incubated in a humid chamber overnight, dried, and washed in PBS and water containing Photoflo (Kodak, NY, USA). Following blocking in 10% donkey serum and 3% BSA, immunofluorescence staining was performed by incubating with primary antibodies: γH2AX (1:500; abcam) and SYCP3 (1:100; Abcam) overnight at room temperature. Alexa 488 donkey anti-rabbit (1:500, Molecular Probes), Alexa 594 goat anti-mouse (1:200, Molecular Probes) were used as secondary antibodies. Slides were incubated with secondary antibodies at 37 °C for 1 hour in the dark, washed and mounted with Vecta shield cover slips. (Vector Laboratories).
7
Testicular Immunohistochemistry Protocol
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Immunohistochemistry analysis of testicular sections was performed using 4% paraformaldehyde fixed tissues as previously described65 (link). Primary antibodies were diluted at 1:50 (GATA1; Santa Cruz Biotechnology sc265), 1:500 (PLZF; R&D Systems AF2944), 1:500 (SYCP3; Abcam ab97672), and 1:200 (STAR; CST 8449 and αSMA; Sigma A2547). Biotinylated polyclonal goat anti-rabbit (Dako, E0432) and anti-mouse (Dako, E0433) antibodies were diluted 1:500. Histological sections were examined using an AxioImager microscope equipped with an AxioCam ICc1 camera and ZEN v.2.3 (Blue edition, Zeiss).
8
Spermatocyte Spreading and Immunofluorescence
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Spreads of spermatocytes and immunofluorescence staining were performed as previously described [25] . In brief, testes of these PBS/DEP-exposed mice were placed in hypotonic extracts (50mM sucrose, 17mM sodium citrate, 30mM Tris in pH8.2, 2.5mm DTT, 1mM PMSF in pH8.3 and 5mM EDTA), incubated on ice for 20 minutes and minced in 100mM sucrose. The spermatocyte spreads were then prepared on a slide and solidified in 1% PFA containing 0.1%
Triton X-100. The slides were incubated overnight in a damp room, dried and washed in PBS and water containing Photoflo (Kodak, NY, USA). Samples were first blocked using 10% donkey serum with 3% BSA, and then stained with primary antibodies γH2AX (1:500, Abcam), SYCP3
Triton X-100. The slides were incubated overnight in a damp room, dried and washed in PBS and water containing Photoflo (Kodak, NY, USA). Samples were first blocked using 10% donkey serum with 3% BSA, and then stained with primary antibodies γH2AX (1:500, Abcam), SYCP3
9
Protein Extraction and Western Blot Analysis
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All of protein was extracted from tissue using a TriPure Isolation Reagent (Roche, Germany). 20 µg of the extracted proteins were loaded on 12% SDS-polyacrylamide gels (BioRad Laboratories, Hercules, CA) and then transferred onto polyvinylidene di uoride (PVDF) membranes (Roche, Germany). The nonspeci c binding was blocked by PBS buffer containing 0.1% Tween-20, 2% BSA, and 5% nonfat dry milk (Carnation, CA). Afterward, samples were incubated by primary antibodies against Id4 (1:1000, Invitrogen, US), Sycp3 (1:1000, Abcam, UK) and β-actin (1:3000, Abcam, UK) as internal control for an overnight at 4°C, and then incubated using secondary antibodies goat anti-mouse IgG H&L-HRP conjugate (1:5000, Bio-Rad, US) and HRP-conjugated goat anti-rabbit IgG H&L (Abcam, USA). Finally, rates of expression for these proteins were evaluated using enhanced chemiluminescence.
10
Meiotic Chromosome Spread Preparation
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Testes of mice were removed and placed into hypotonic extracts [50mM sucrose, 17mM sodium citrate, 30mM Tris (pH 8.2), 2.5mM DTT, 1mM PMSF (pH8.3) and 5mM EDTA] after the tunica albuginea was removed, then incubated on ice for 20 min and minced in 100mM sucrose. The spermatocyte spreads were prepared on a slide and solidi ed in 1% paraformaldehyde containing 0.1% Triton X-100. The slides were incubated for 6 h in a damp room and washed with 0.04% Photo-Flo (Kodak, NY, USA). After that, the slides were dried and blocked 30 min using 10% donkey serum with 3% bovine albumin and incubated overnight with the primary antibody [γH2AX (1:500), SYCP3 (1:100) or DMC1 (1:100)] (Abcam, Cambridge, MA, USA) at 37℃, followed by incubated with secondary antibody in darkness for 1 h at 37℃. After washing, drying and mounting with Vecta shield cover slips (Vector Labs, Burlingame, CA, USA), the slides were observed and photographed using super-resolution microscope (Nikon N-STORM, Tokyo, Japan).
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