The prepared sections were blocked with 3% (w/v) bovine serum albumin (BSA; ZSbio) in PBST (0.1% [v/v] Triton X-100 in PBS) for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4°C: anti-Sycp3 (1:200, Abcam, Cambridge, MA, USA), anti-synaptonemal complex protein 1 (Sycp1; 1:200, Abcam), anti-Vasa (1:500, Abcam), anti-c-kit (1:500, Abcam), anti-Shp2 (1:200, Santa Cruz Biotechnology), anti-Plzf (1:500, Santa Cruz Biotechnology), anti-cleaved caspase 3 (1:200, Cell Signaling Technology, Boston, MA, USA), anti-Dmc1 (1:200, Abcam), anti-Smc3 (1:500, Abcam), and anti-DNA repair recombinase rad51 (Rad51; 1:200, Invitrogen). After being washed three times with PBST, the samples were incubated with the following secondary antibodies at a 1:200 dilution for 1 h at 37°C: Alexa Fluor 594/488-labeled anti-rabbit or anti-mouse IgG (YEASEN, Shanghai, China). The slides were subsequently washed three times in PBST and mounted with Vectashield containing 4’-6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA).
Anti sycp3
Manufactured by Abcam
Sourced in United States
Anti-SYCP3 is a primary antibody that specifically binds to the SYCP3 (Synaptonemal Complex Protein 3) protein. SYCP3 is a structural component of the synaptonemal complex, which is essential for chromosome pairing and segregation during meiosis. This antibody can be used for applications such as immunohistochemistry and Western blotting to detect and study the SYCP3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (2 mentions)
Fitc conjugated goat anti mouse, by Jackson ImmunoResearch (2 mentions)
Anti γh2ax, by Merck Group (2 mentions)
Anti γh2ax antibody, by Merck Group (2 mentions)
Anti plzf, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin bsa, by ZSGB-BIO (1 mentions)
Sycp1, by Abcam (1 mentions)
Anti vasa, by Abcam (1 mentions)
Anti c kit, by Abcam (1 mentions)
Anti shp2, by Santa Cruz Biotechnology (1 mentions)
Anti dmc1, by Abcam (1 mentions)
Anti smc3, by Abcam (1 mentions)
Vectashield containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Isis4 image processing package, by MetaSystems (1 mentions)
Anti cdk2, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
8 protocols using anti sycp3
1
Immunofluorescence Staining of Germ Cell Markers
2
Immunostaining and FISH Analysis of Meiotic Cells
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The spreads of meiotic cells were prepared according to the protocol of Peters et al. (1997) (link). Immunostaining was performed according to the protocol described by Anderson et al. (1999) (link) using rabbit polyclonal anti-SYCP3 (1:500, Abcam), mouse monoclonal anti-MLH1 (1:50, Abcam), and human anticentromere (ACA ) (1:100, Antibodies Inc) primary antibodies. As secondary antibodies Cy3-conjugated goat anti-rabbit (1:500, Jackson ImmunoResearch), FITC -conjugated goat anti-mouse (1:50, Jackson ImmunoResearch), FITC -conjugated donkey anti-human (1:100, Vector Laboratories) were used. All antibodies were diluted in PBT (3% bovine serum albumin and 0.05% Tween 20 in 1xPBS). A solution of 10% PBT was used for blocking non-specific antibody binding. Primary antibody incubation was performed overnight in a humid chamber at 37°C, and secondary antibody incubation was performed for 1 h at 37°C. Finally, slides were mounted in Vectashield with DAPI (Vector Laboratories) to stain DNA and reduce fluorescence fading. After image acquisition of the immunofluorescent signals, the slides were subjected to FISH .
The preparations were visualized with an Axioplan 2 Imaging microscope (Carl Zeiss) equipped with a CCD camera (CV M300, JAI), CHROMA filter sets, and ISIS4 image processing package (MetaSystems GmbH).
The preparations were visualized with an Axioplan 2 Imaging microscope (Carl Zeiss) equipped with a CCD camera (CV M300, JAI), CHROMA filter sets, and ISIS4 image processing package (MetaSystems GmbH).
3
Chromosome Spread Preparation and Immunostaining
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Prophase I chromosome spreads from adult testes were prepared as previously described [28 (link),61 (link)]. For all experiments, at least 6 males of each genotyped were evaluated. Chromosome slides were then washed in 0.4% Kodak Photo-Flo 200/1X PBS for 2 x 5 min, 0.4% Kodak Photo-Flo 200/dH2O for 2 x 5 min, then air-dried for approximately 10 min and stored in -80°C or used immediately for staining. Primary antibodies used were: anti-γH2AX (Millipore, NY, #05–636 1:10,000), anti-SYCP3 (Abcam, MA, #97672, 1:5000), anti-SYCP1 (Abcam, MA, #15087, 1:1000), anti-RAD51 (Calbiochem, #PC130, 1:500), anti-BLM (generous gift from Dr. Ramundo Freire; 1:100;), anti-CDK2 (Santa Cruz, TX, sc-163; 1:250), anti-MLH3 ([61 (link)]; 1:1000), anti-RNF212 (generous gift from Dr. Neil Hunter), anti-RPA (generous gift from Dr. Jeremy Wang; 1:500), anti-MSH4 (Abcam, MA, #58666; 1:500), anti-HEI10 (Anti-CCNB1IP1, Abcam, MA # 71977) and anti-MLH1 (BD Biosciences Pharmingen, CA, #550838, 1:100). Secondary antibodies used were: goat anti-mouse Alexa Fluor 488 (#62–6511), goat anti-mouse Alexa Fluor 555 (#A-10521), goat anti-rabbit Alexa Fluor 488 (#65–6111), goat anti-rabbit Alexa Fluor 555 (#A-10520; all Invitrogen, 1:2000).
4
Immunofluorescence Staining and Pulldown Assay
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Anti-RAD51 (1:100 Santa Cruz, sc8364), anti-DMC1 (1:50 Santa Cruz, sc22768), anti-SYCP3 (1:500 abcam, ab97672), anti-γH2AX (1:500 Millipore, 05–636) antibodies were used for immunofluorescence staining of spermatocyte spread. Anti-RAD51 (1:500 Millipore, ABE257) and anti-γH2AX (1:1000 Millipore, 05–636) antibodies were used for immunofluorescence staining of U2OS cells. Anti-FLAG (1:3000 Wako 012-22384) and anti-Myc (1:3000 Nacalai 04362-34) antibodies were purchased from Wako and Nacalai, respectively. For pulldown assay, anti-FIGNL1 (1:500 abcam, ab173685) and anit-SWSAP1 (1:500 Thermo, PA5-25460) were used.
5
Antibody Sources for TDP-43, Stra8, SYCP3, and More
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Guinea pig anti-TDP-43 polyclonal antibody was reported previously (15 (link)). Antibody to Stra8 was a kind gift from Dr Michael Griswold, Washington State University. Anti-SYCP3 (Abcam; 15093), anti-γH2AX antibody (Millipore Sigma; 05-636), and anti-Sox9 antibody (Millipore; AB5535) were obtained from commercial sources. anti-γH2AX antibody used for IHC was a mouse monoclonal from Millipore 05-636. Guinea pig anti-SP-10 polyclonal antibody was reported previously (63 (link)).
6
Immunofluorescence Staining of Dazl and Vasa
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For immuno-staining of Dazl protein, cells were fixed with 4% paraformaldehyde in PBS for 1 hr at room temperature, permeabilized with 0.3% Triton-X in PBS for 15 min. For immuno-staining of Vasa protein, cells were fixed with 4% paraformaldehyde in PBS for 1 hr at room temperature, incubated with 0.2 M Glycine for 10 min at room temperature. Then the cells were re-fixed with 100% methanols for 10 min at −20 °C, and were incubated with blocking solution (10% FBS, 1% BSA, 0.1% Triton-X in PBS) for 1 hr at 4 °C. The cells were then incubated with primary antibody (rabbit anti-Dazl, Abcam; rabbit anti-Vasa49 ) in the blocking solution overnight at 4 °C. The cells were then washed and incubated with secondary antibody (Alexa Fluor dye-conjugated secondary antibodies, Invitrogen) and 3 μg/ml−1 4′,6-diamidino-2-phenylindole (DAPI) in blocking solution for 1 hr at room temperature. The cells were washed again and mounted with VECTASHIELD (Vectro Laboratories). Stained cells were observed under a Leica AF6000 fluorescence microscope, and were then analyzed with image J. The immune-staining of Sycp3 protein were performed as described previously using anti-Sycp3 (Abcam)40 .
7
Chromosome Spreading of Human Spermatocytes
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Chromosome spreading of primary human spermatocytes was performed using the drying-down technique [15 (link)]. Briefly, human seminiferous tubules were isolated. Subsequently, the seminiferous tubules were torn into small pieces. The spermatocyte cell suspension was mixed with 3.7% PFA solution. The spermatocyte cells were subsequently spread on a clean glass slide. Then, the cell slides were washed, dried, and stained using anti-SYCP3 (1:200, Abcam), γ-H2AX (1:200, Sigma), and/or anti-Fem9b (1:200, Abcam). TRITC- or FITC-conjugated anti-rabbit secondary antibody (Jackson Laboratories, Bar Harbor, ME)) was applied at a dilution of 1:600. Protein subcellular localizations were examined using a laser confocal microscope (Zeiss).
8
Immunostaining of Meiotic Chromosomes
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Chromosome spreads were prepared by the drying-down method [Peters et al., 1997] . Immunostaining was performed according to Anderson et al. [1999] using rabbit polyclonal anti-SYCP3 (1:500; Abcam, cat. No. ab15093), human anticentromere (1:100; Antibodies Inc., cat. No. 15-234), and mouse monoclonal anti-H3K9me2/3 (1:100, Cell Signaling, cat. No. 5327) primary antibodies. The secondary antibodies used were Cy3-conjugated goat anti-rabbit (1:500; Jackson ImmunoResearch, , AMCA-conjugated donkey anti-human (1:100; Jackson , and FITC-conjugated goat anti-mouse (1:100; Jackson ImmunoResearch, cat. No. 115-095-003). The slides were incubated overnight with primary antibodies and for 1 h with secondary antibodies at 37°C in a humid chamber. Slides were mounted in Vectashield antifade mounting medium (Vector Laboratories, USA, cat. No. H-1000-10).
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