Testes were fixed at 4° C in Bouins solution, and dehydrated for paraffin embedding. Sections were cut to a thickness of 5 μM, deparaffinized and rehydrated. For histology, sections were stained with hematoxylin and eosin (H+E). For immunohistochemistry, slides were prepared by performing antigen retrieval. Slides were boiled in 0.01 M sodium citrate, pH 6.0, for 10 min. Sections were then blocked for 1 h at room temperature with 3% goat serum in PBS. Primary antibodies were diluted in 3% goat serum in PBS and incubated with sections overnight at 4° C. Primary antibodies used for IHC were anti-SYCP3 (#ab15093, Abcam, Cambridge, MA), anti-γH2AX (#05-636, Millipore, Billerica, MA), anti-STRA8 (#ab49602, Abcam), anti-MED1 (#ab64965, Abcam), anti-TRA98 (#ab82527, Abcam), anti-BOULE (gift of Eugene Xu, Northwestern University), anti-SOHLH1 (gift of Aleksandar Rajkovic, University of Pittsburgh). Sections were then labeled with the appropriate fluorescence-conjugated secondary antibody for 1 h at room temperature. Slides were incubated for 10 min with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were then affixed with Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA) and samples were imaged using a Leica DMR-HC epifluorescence microscope with 10x, 20x, and 40x objectives and a QImaging Retiga 4000R camera.
Ab15093
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab15093 is a primary antibody produced in rabbit. It recognizes the antigen of interest. The core function of this product is to bind and detect the target protein in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab15090, by Abcam (7 mentions)
Ab13840, by Abcam (7 mentions)
Ab97672, by Abcam (6 mentions)
Ab49602, by Abcam (5 mentions)
Sc 28319, by Abcam (3 mentions)
Ab14206, by Abcam (3 mentions)
Ab26350, by Abcam (3 mentions)
Vectashield, by Vector Laboratories (3 mentions)
Ab49405, by Abcam (3 mentions)
Ab2893, by Abcam (3 mentions)
Ab5535, by Merck Group (3 mentions)
Ab82527, by Abcam (2 mentions)
Ab189849, by Abcam (2 mentions)
Ab150077, by Abcam (2 mentions)
Dm400bled368424, by Leica camera (2 mentions)
Ab15580, by Abcam (2 mentions)
Ab15087, by Abcam (2 mentions)
Ab13939, by Abcam (2 mentions)
Confocal laser scanning microscope, by Zeiss (2 mentions)
Sc 74569, by Santa Cruz Biotechnology (2 mentions)
44 protocols using ab15093
1
Immunohistochemical Analysis of Testis
2
Meiotic Progression in Tetraploid Cells
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Meiosis spread assay was performed to determine the meiosis progression in the FACS sorted tetraploid cells. Briefly, 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 hours at room temperature in a humid chamber. The cells were treated with 0.04% photoflo for 5 min and blocked with 4% goat serum. Staining was performed in cells incubated with primary antibodies anti-SCP3 (ab15093, Abcam) overnight at 37 °C in a humid chamber. Donkey Anti-Rabbit IgG (H + L)(Alexa Fluor 555) (A31572, Molecular Probes) were applied at 1:1000 dilution and incubated for 90 min at 37 °C. Cells were washed three times with PBS and counterstained with DAPI, and images were captured with a fluorescence microscope.
3
Meiotic Proteins Immunofluorescence and Western Blot
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The following antibodies were purchased: anti-SYCP3 (Abcam, ab97672, IF: 1:100), anti-SYCP3 (Abcam, ab15093, IF: 1:100), anti-SYCP1 (Abcam, ab15090, IF: 1:100), anti-γH2AX (MerckMillipore, 16-202A, IF: 1:800), anti-TNP2 (Santa Cruz, sc-393843, IF: 1:50), anti-PRM2 (Briar Patch Biosciences, Hup 2B, IF: 1:300), anti-H4K8ac (ABclonal, A7258, IF: 1:500), anti-ELFN2 (Novus Biologicals, 90569, WB: 1: 1000), anti-H3 (Abcam, ab1791, WB: 1: 2000), anti-GAPDH (Abcam, ab8245, WB: 1: 10000), anti-TUBLIN (Sigma, T8328, WB: 1: 2000), anti-FLAG (Sigma, F3165, IP: 10 µg, WB: 1:2000). For immunofluorescence studies, the following secondary antibodies were used: anti-rabbit (Jackson ImmunoResearch 488, 711225152, IF: 1:350), anti-rabbit (Jackson ImmunoResearch 594, 711585152, IF: 1:350), anti-mouse (Jackson ImmunoResearch 488, 715545150, IF: 1:350). For Western blot analyses, the following secondary antibodies conjugated to Horse Radish Peroxidase were used: anti-rabbit IgG HRP-linked (ABclonal, AS014, WB: 1:5000), anti-mouse IgG HRP-linked (ABclonal, AS003, WB: 1:5000).
4
Meiotic DNA Spreading Assay Protocol
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Meiotic DNA spreading assays were performed according to published protocols (Durruthy Durruthy et al., 2014 (link), Kee et al., 2009 (link)) with minor modifications. In brief, the dissociated cells were suspended in hypo-extraction buffer (30 mM Tris, 50 mM sucrose, 17 mM Na citrate, and 5 mM EDTA, pH 8.3, Protease Inhibitor Cocktail/Santa Cruz Tech) and incubated on ice for 30 min. Following centrifugation, the cells were resuspended in 20 μL hypoextraction buffer with 60 μL 100 mM sucrose and cytospun at 180 × g for 3 min onto a slide. The cells were fixed in 4% PFA, and incubated with 0.04% photoflo (Kodak) for 5 min. Air-dried slides were blocked with 3% BSA for 30 min at room temperature and stained with SYCP3 (ab15093) and CENPA (ab13939) antibodies (both from Abcam) at 4°C overnight, and with fluorescein-conjugated secondary antibodies thereafter. Images were captured with a Leica confocal microscope.
5
Meiotic Chromosome Spreads Immunofluorescence
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Spermatocyte spreads were prepared as previously described by Peters et al. (1997) (link). Primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam, ab97672), rabbit anti-SYCP1 (C-terminal) (1:2,000 dilution; Abcam, ab15090), Anti-phospho-Histone H2A.X (Ser139) Antibody (1:1,000 dilution; Millipore, 05-636), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology, sc-22768), rabbit anti-RAD21(1:100 dilution; Abclonal, A18850), rabbit anti-RAD51 polyclonal antibody (1:200 dilution; Thermo Fisher Scientific, PA5-27195) Primary antibody incubated overnight at four degrees in the refrigerator and were detected with Alexa Fluor 488- or 594-conjugated secondary antibodies for 1 h at room temperature. After being washed with PBS several times, the slides were mounted using Mounting medium with DAPI-aqueous, fluoroshield (Abcam, ab104139).
6
Comprehensive Protein Analysis in Spermatogenesis
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Western blot analysis was done as described previously.14 The antibodies used in IHC were also applied to the Western blotting analysis. Primary antibodies were raised against proteins associated with spermatogonia proliferation (PCNA, ab92552; PLZF, ab189849, Abcam), meiosis (SYCP3, ab15093; STRA8, ab49602, Abcam; REC8, D222997; MLH1, D121003; DMC1, D224646, BBI Solutions), the blood‐testis barrier (β‐catenin, ab32572; ZO‐1, ab96587, Abcam), an apoptosis‐associated protein (Bax, ab32503) and sperm quality (PGK2, ab183031; HSPA4L, ab87241, Abcam).
7
Immunofluorescence Staining of Cell Cultures
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The cells were seeded on coverslips and the fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were washed with PBS once and subsequently soaked in the mixing buffer of equal volume of 0.1% Triton X-100 and 3% BSA for 1 h at room temperature. The cells were washed with PBS once, then incubated with primary antibody at 4 °C for overnight. The cells were washed with PBS five times next day, then incubated with appropriate secondary antibody for 1 h at room temperature. The cells were washed with PBS five times to remove the secondary antibody and counterstained with DAPI for 3 min and washed with PBS once. In the end, the coverslips were mounted on the slide for observation under the confocal microscope (Zeiss 710 NLO). Primary antibodies and secondary antibodies were diluted in 3% BSA. Primary antibodies used in this study were anti-KLF4 (R&D system, AF3158, 1:100), anti-H3K9me2 (Abcam, ab1220, 1:100), anti-H3K27me3 (Merck, 17–622, 1:200), anti-PRDM1 (Invitrogen, 14–5963–82, 1:50), anti-DDX4 (Abcam, ab13840, 1:400), anti-SOX9 (EMD Millipore, AB5535, 1:400), anti-PLZF (Santa Cruz, sc-28319, 1:100), anti-SYCP3 (Abcam, ab15093, 1:400), and anti-γH2AX (Abcam, ab26350, 1:400), anti-PNA (ThermoFisher, L21409, 10 μg/ml).
8
Immunofluorescence Analysis of Meiotic Chromosomes
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Spermatocyte and oocyte chromosome spreading was prepared as previously described (50 (link), 51 (link)). Structurally preserved spermatocytes were prepared as described previously (52 (link)).
Primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam #ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-SYCP1 (C-terminal) (1:2000 dilution; Abcam #ab15090), rabbit anti-SYCP1 (N-terminal) (1:2000 dilution; Abclonal #A12139), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology #sc-22768), mouse anti–phospho-histone H2AX (pSer139) (1:300 dilution; Millipore #05-636), mouse anti-MLH1 (1:50 dilution; BD Biosciences #550838), rabbit anti-TEX12 (1:1000 dilution; Proteintech #17068-1-AP), mouse anti-TRF1 (1:1000 dilution; homemade), and rabbit anti-BRCA1 (1:500 dilution; a gift from L.-Y. Lu, Zhejiang University). Primary antibodies were detected with Alexa Fluor 488– or 594–conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, and #ab150120) for 1 hour at room temperature. The slides were washed several times with PBS and mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H-1200).
Primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam #ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-SYCP1 (C-terminal) (1:2000 dilution; Abcam #ab15090), rabbit anti-SYCP1 (N-terminal) (1:2000 dilution; Abclonal #A12139), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology #sc-22768), mouse anti–phospho-histone H2AX (pSer139) (1:300 dilution; Millipore #05-636), mouse anti-MLH1 (1:50 dilution; BD Biosciences #550838), rabbit anti-TEX12 (1:1000 dilution; Proteintech #17068-1-AP), mouse anti-TRF1 (1:1000 dilution; homemade), and rabbit anti-BRCA1 (1:500 dilution; a gift from L.-Y. Lu, Zhejiang University). Primary antibodies were detected with Alexa Fluor 488– or 594–conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, and #ab150120) for 1 hour at room temperature. The slides were washed several times with PBS and mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H-1200).
9
Immunofluorescence Characterization of Germ Cell and Oocyte Markers
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We used 4% paraformaldehyde/PBS at room temperature for 45 minutes to fix cells and permeabilization of cells was done by 0.2% Triton X-100 in PBS. The cells were blocked overnight with a blocking solution (5% milk and 0.05% Tween-20 in PBS) and then incubated with primary antibodies by using 1:100 dilution of monoclonal anti-DDX4 (ab13840, Abcam, USA), monoclonal anti-DAZL (ab34139, Abcam, USA), as a germ cell markers and monoclonal anti-SCP3 (ab15093, Abcam, Cambridge, UK), monoclonal anti-GDF9(ab93892, Abcam, USA) and monoclonal anti-GDF9B (ab108413, Abcam, Cambridge, UK) as oocyte like cells markers for 2.5 hrs. Afterward, cells were washed in PBS/Tween 20 (0.1%) (Sigma) three times and incubated with Alexa fluor 488 and 594 secondary antibodies (Sigma, USA) for 1hrs in a dark place at room temperature. Cells were again washed with PBS/Tween 20 (0.1%) three times. Finally, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D8417) and studied by Olympus DP73 digital camera associated with a fluorescence microscopy IX81 (U-MW-IB3). ImageJ software was used to assess the proportion of DDX4, DAZL, SCP3, GDF9, and GDF9B proteins in the resultant microphotographs.
10
Immunofluorescence Staining of Germ Cells
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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).
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