Germline antibody staining was performed as previously described [62 (link)]. Briefly, dissected gonads were fixed in 3% paraformaldehyde/0.1M K2HPO4 (pH 7.2) solution for 10-20 min, and then post-fixed with cold 100% methanol for 5 min. After blocking for 30 min in 1× PTW (1×PBS + 0.1% Tween20)/0.5% BSA (Bovine Serum Albumin) solution, primary antibody was added, followed by incubation for 2 h at 20°C. The dissected gonads were washed three times for at least 30 min with 1× PTW/0.5% BSA solution and incubated in the same solution containing the fluorescence-conjugated secondary antibodies for 1-2 h at 20°C. After washing three times in 1× PTW/0.5% BSA solution for at least 30 min, the dissected gonads were stained with 100 ng/mL DAPI solution for 10 min at 20°C and were again washed in 1× PTW/0.5% BSA solution three times. The antibody staining was observed using fluorescence microscopy. Antibodies used in this study include anti-REC-8 (1:200 dilution, kindly provided by Dr. Josef Loidl's lab, University of Vienna, Austria), anti-HIM-3 (1:400 dilution, Novus Biologicals, Littleton, CO, USA), anti-GFP (1:400 dilution, Abcam, Cambridge, MA, USA), and anti-GLP-1 (1:200 dilution, kindly provided by Dr. Judith Kimble's lab, University of Wisconsin-Madison, WI, USA).
Anti him 3
Manufactured by Novus Biologicals
Sourced in United States
Anti-HIM-3 is a polyclonal antibody developed for the detection of HIM-3 protein. HIM-3 is a meiosis-specific protein involved in chromosomal synapsis and recombination during meiosis. The antibody can be used in various applications such as Western blotting, immunohistochemistry, and immunofluorescence to study the expression and localization of HIM-3 in biological samples.
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Lab products found in correlation
3 protocols using anti him 3
1
Germline Antibody Staining Protocol
2
Germline Antibody Staining Protocol
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Germline antibody staining was performed as previously described (Yoon et al., 2016 (link)). Briefly, dissected gonads were fixed in 3% paraformaldehyde/0.1M K2HPO4 (pH 7.2) solution for 10–20 min, and then post-fixed with cold 100% methanol for 5 min. After blocking for 30 min in 1× PBST (1XPBS + 0.1% Tween 20)/0.5% BSA (Bovine Serum Albumin) solution, primary antibody was added, followed by incubation for 2 h at 20°C (or overnight at 4°C). The dissected gonads were washed three times for at least 30 min with 1× PBST/0.5% BSA solution and incubated in the same solution containing the fluorescence-conjugated secondary antibodies for 1–2 h at 20°C. After washing three times in 1× PBST/0.5% BSA solution for at least 30 min, the dissected gonads were stained with 100 ng/mL DAPI solution for 10 min at 20°C and were again washed in 1× PBST/0.5% BSA solution three times. The antibody staining was observed using fluorescence microscopy. Primary antibodies used in this study include anti-HIM-3 (Novus Biologicals, 1:400 dilution), anti-GLD-1 (provided by Dr. Kimble’s Lab, 1:200 dilution), anti-MSP (Developmental Studies Hybridoma Bank – University of Iowa), and anti-GFP (Abcam, Cambridge, MA, United States, 1:400 dilution). Alexa 488- or CY3-conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA, United States, 1:200 dilution) were diluted to 1:300.
3
Single-Molecule Localization Microscopy Sample Preparation
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For SMLM, coverslips (Precision coverslips, 24 mm, Carl Roth) were cleaned by a 10-min wash in ethanol, rinsed in milli-Q water, and plasma cleaned (PlasmaPrep2, GaLa Instrumente). Clean slides were incubated with 0.01% poly-l -lysine (30,000–70,000 D, Sigma-Aldrich) for 10 min. After incubation, coverslips were rinsed in milli-Q water and dried at room temperature. Poly-l -lysine–coated coverslips were stored at 4°C until use.
Immunofluorescence samples for SMLM imaging were prepared as described above with a few modifications. Worms were immobilized in 0.02% of tetramisole during dissection and fixed with 1% PFA on poly-l -lysine–coated coverslips. Primary antibodies used for SMLM were: anti-HA (1:200, mouse monoclonal 2-2.2.14, Thermo Fisher Scientific), anti-HIM-3 (1:200 or 1:100, rabbit polyclonal 53470002, Novus Biologicals). Secondary antibodies were Alexa Fluor 647 anti-mouse (1:1,000 or 1:500, donkey polyclonal, Jackson Immunoresearch) and CF680 anti-rabbit (1:1,000 or 1:500, goat F(ab)′ fragment, Sigma-Aldrich). The samples were mounted in a custom sample holder and imaged in blinking buffer (50 mM Tris HCl, pH 8, 10 mM NaCl, 10% (wt/vol) d -glucose, 500 µg/ml glucose oxidase, 40 µg/ml catalase, and 35 mM mercaptoethylamine).
Immunofluorescence samples for SMLM imaging were prepared as described above with a few modifications. Worms were immobilized in 0.02% of tetramisole during dissection and fixed with 1% PFA on poly-
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