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.
Ab49602
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab49602 is an anti-Glutamic acid decarboxylase 1 (GAD1) antibody. It is a polyclonal antibody produced in rabbit and purified by protein A affinity chromatography. The antibody is designed for the detection of GAD1 protein in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ab15093, by Abcam (5 mentions)
Ab13840, by Abcam (5 mentions)
Ab82527, by Abcam (3 mentions)
Eclipse te2000 s microscope, by Nikon (2 mentions)
Ab189849, by Abcam (2 mentions)
Fl 335, by Santa Cruz Biotechnology (2 mentions)
A1978, by Merck Group (2 mentions)
Ab97672, by Abcam (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab64965, by Abcam (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Ab290, by Abcam (1 mentions)
Af2030, by R&D Systems (1 mentions)
Alexa fluor 488 donkey anti chicken, by Jackson ImmunoResearch (1 mentions)
Nb300 230, by Novus Biologicals (1 mentions)
Alexa fluor 546 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
Ab183031, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
21 protocols using ab49602
1
Immunohistochemical Analysis of Testis
2
Immunohistochemical Analysis of Germ Cell Markers
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Freshly collected tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for analysis using primary antibodies against Stra8 (rabbit polyclonal, ab49602; Abcam), Ddx4 (rabbit polyclonal ab13840, Abcam; goat polyclonal AF2030, R&D Systems), Sycp3 (rabbit polyclonal NB300-230, Novus Biologicals), Ser139-phospho-H2afx (mouse monoclonal 05–636, Millipore) or GFP (chicken polyclonal ab13970, Abcam; rabbit polyclonal ab290, Abcam). For IF, detection was performed using donkey anti-chicken Alexa Fluor 488 (Jackson Immuno), donkey anti-goat Alexa Fluor 647 or donkey anti-rabbit Alexa Fluor 546 (Molecular Probes) as secondary antibody16 (link). For IHC, detection was performed using biotin-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) as secondary antibody for horseradish peroxidase-based DAB detection (Sigma-Aldrich). Images were captured using a Nikon E800/BioRad Radiance 2000 confocal microscope or a Nikon ECLIPSE TE2000-S microscope.
3
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).
4
Immunostaining of Testicular Cell Markers
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Briefly, the testis was fixed with 4% paraformaldehyde, followed by wax dipping, dehydration, and fluorescent staining (anti-STRA8, 1:200, Abcam, ab49602; anti-TRA98, 1:200, Abcam, ab82527; anti-SYCP3, 1:200, Abcam, ab97672; anti-E2F2, 1:200, Affinity, AF4100). Immunofluorescence and HE staining steps were as previously described [50 (link), 51 (link)].
5
Western Blot Analysis of Protein Expression
6
Immunohistochemical Characterization of HORMAD1 and STRA8
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Immunohistochemistry was performed as previously described [9 (link),37 (link)]. Samples were incubated with HORMAD1 rabbit polyclonal antibody (Novus Biologicals, Centennial, CO, USA, NBP1-85401) or STRA8 (Abcam, ab49602; RRID:AB_945678) at a dilution of 1:500. Immunoreactivity was evaluated in Quantitative Pathology and Bioimage Analysis (Qupath; RRID:SCR_018257) using images of samples scanned on a Zeiss Axio scanner. Cell counts were performed randomly in three regions. The percentage of HORMAD1 or STRA8 immunostaining was used for analysis across 18 samples with corresponding positive and negative controls.
7
Multimarker Immunofluorescence Staining
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The antibody used in immunofluorescence staining were as follows: anti-DDX4 (ab13840, Abcam), anti-GATA4 (ab124265 Abcam), anti-c-KIT (25-1171-82, eBioscience) anti-SYCP3 (ab97672, Abcam), anti-STRA8 (ab49602, Abcam), anti-γH2AX (ab2893, Abcam), anti-H3pSer10 (ab5176, Abcam), anti-MPS1 (ab11108, Abcam), anti-H2BK120ubi (#5546, CST), anti-MDM2 (sc-965, Santa Cruz), anti H2B (ab1790, Abcam), and anti-ACTIN (sc-47778, Santa Cruz). All fluorophore-conjugated secondary antibodies were obtained from Jackson Immuno Research.
8
Western Blot Analysis of Protein Extracts
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The total protein was extracted in the extraction buffer [20% glycerol, 50 mmol/L Tris-HCl (pH 6.8) and 0.5% (v/v) Tween 20], to which a protease inhibitor cocktail (Sigma-Aldrich, US) was added. Next, the samples were centrifuged at 12,000 rpm (6439 g) for 10 min to remove nondissolved material. The protein concentration in the supernatant was measured using a Tiangen protein assay kit (Tiangen Biotech, China). Aliquots of protein extract containing 30 µg of total protein were electrophoresed in a 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a P-membrane (Millipore, US) for 3 h with transfer buffer (25 mmol/L Tris, 192 mmol/L glycine, and 20% (v/v) methanol). The membrane was blocked with 2% dried nonfat milk and incubated with antibodies against L19 (sc-100830, Santa Cruz, US, 1∶500) and Stra8 (ab49602 Abcam, US, 1∶500). After treatment with the primary antibody, the membrane was washed in Tris-buffered saline-Tween buffer (20 mmol/L Tris, 500 mmol/L NaCl, 0.05% Tween 20) and incubated with the secondary antibody (dilution at 1∶3000). Final exposure was achieved using enzymatic chemiluminescence (Amersham Pharmacia, UK). The films were scanned and quantified using ImageJ software (n = 6/group).
9
Immunostaining of Germ Cell Markers
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Cells underwent a 15 min fixation in 4% paraformaldehyde (PFA) at room temperature (RT), followed by a 20 min blocking in 0.3% Triton X-100/2% BSA in PBS, and subsequent overnight (ON) 4 °C incubation in primary antibodies against OCT4 (1:200,P0056, Merck, Germany), PLZF (1:200, ab305064, Abcam), GFP (1:200 Millipore, Merck, Darmstadt, Germany), DDX4 (1:500 Abcam, ab27591, Minneapolis, MI, USA), and STRA8 (1:200 Abcam, ab49602, Minneapolis, MI, USA). The samples were then thrice PBS-rinsed and then exposed for 2 h to fluorescein isothiocyanate (FITC)-labeled secondary antibodies, Alexa Fluor 555, and Alexa Fluor 647 (Thermo Fisher Scientific, Waltham, MA, USA). The DNA underwent a 10 min counterstaining in 10 mg/mL Hoechst 33,342 (Thermo Fisher Scientific, Waltham, MA, USA), with three subsequent PBS rinses. Lastly, image capture was performed using a Zeiss LSM800 Meta inverted confocal microscope.
10
Protein Expression Analysis in Testes
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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).
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