Sperm specimens were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 10 min. Non-specific sites were blocked with 10% normal goat serum and 3% bovine serum albumin in phosphate-buffered saline, and the samples were incubated overnight at 4 °C with monoclonal anti-acetylated α-tubulin (T5168, 1:800), SPAG6 (HPA038440, 1:400), polyclonal anti- RSPH1 (HPA017382, 1:400), and polyclonal anti-AKAP4 (HPA020046, 1:200) antibodies (all from Sigma-Aldrich, St. Louis, MO, USA); and polyclonal antibodies against DNAH6 (ab122333, 1:50) and DNAH1 (ab122367, 1:100) (both from Abcam, Cambridge, UK). Alexa Fluor 488 anti-mouse (A-21121, 1:300) and Alexa Fluor 555 anti-rabbit (A31572, 1:300 dilution) IgG (both from Life Technologies, Carlsbad, CA, USA) were used as secondary antibodies. Specimens were counterstained with 4′,6-diamidino-2-phenylindole for 5 min, and fluorescence signals were visualized with a BX-51 fluorescence microscope (Olympus, Tokyo, Japan). Images were captured using VideoTesT-FISH v.2.0 software (VideoTesT, St. Petersburg, Russia).
Ab122333
Manufactured by Abcam
Sourced in United States
Ab122333 is a lab equipment product manufactured by Abcam. It is a specialized tool designed for use in laboratory settings. The core function of this product is to facilitate specific tasks or experiments that are essential for scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab122367, by Abcam (2 mentions)
Hpa038440, by Merck Group (2 mentions)
Hpa017382, by Merck Group (2 mentions)
Hpa020046, by Merck Group (2 mentions)
Bx51 fluorescence microscope, by Olympus (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A21121, by Thermo Fisher Scientific (1 mentions)
T5168, by Merck Group (1 mentions)
T6793, by Merck Group (1 mentions)
Hpa021624, by Merck Group (1 mentions)
Nbp2 14260, by Novus Biologicals (1 mentions)
Alexa fluor 488, by Zeiss (1 mentions)
Bs084, by Biosharp (1 mentions)
Alexa fluor 594, by Zeiss (1 mentions)
Hoechst 33342, by Zeiss (1 mentions)
Lsm 800, by Zeiss (1 mentions)
T8203, by Merck Group (1 mentions)
T6199, by Merck Group (1 mentions)
4 protocols using ab122333
1
Immunofluorescence Analysis of Sperm Ultrastructure
2
Sperm Immunofluorescence Microscopy
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Sperm samples were incubated with mouse monoclonal antibody targeting α-tubulin (T5168, Sigma-Aldrich; 1:1,000), rabbit polyclonal antibody targeting DNAH6 (ab122333, Abcam; 1:100), secondary antibodies [Alexa Fluor 488 anti-mouse immunoglobulin G (IgG) (A21121, Life Technologies; 1:1,000) and Alexa Fluor 555 anti-rabbit IgG (A31572, Life Technologies; 1:1,000)], and 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were captured using a BX-51 fluorescence microscope (Olympus, Japan). Images were analyzed using VideoTesT-FISH software (v.2.0, VideoTesT, St. Petersburg, Russia).
3
Sperm Flagellar and Acrosomal Protein Analysis
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A sperm sample from patient AY0283 and two normal sperm samples fixed with 4% PFA were applied to slides, permeabilized with 0.1% Triton X-100 (BS084, Biosharp) for 10 min, and then blocked with 10% donkey serum for 2 h at 37°C. To analyze the location and expression of DNAH6 in spermatozoa and the changes in flagellar-associated and acrosome-associated proteins, anti-DNAH6 (rabbit, 1:100, ab122333, Abcam, Cambridge, UK), anti-DNAH1 (rabbit, 1:100, ab122367, Abcam), anti-DNAI2 (rabbit, 1:200, 17533-1-AP, Proteintech), anti-SPAG6 (rabbit, 1:200, HPA038440, Sigma-Aldrich), anti-RSPH1 (rabbit, 1:100, HPA017382, Sigma-Aldrich), and anti-AKAP4 (rabbit, 1:200, HPA020046, Sigma-Aldrich) antibody were co-incubated with monoclonal anti-acetylated-tubulin antibody (mouse, 1:500, T6793, Sigma-Aldrich) at 4°C for 16 h. Anti-ACTL7A (rabbit, 1:100, HPA021624, Sigma-Aldrich) and anti-acrosin antibodies (rabbit, 1:200, NBP2-14260, Novus) were also incubated separately at 4°C for 16 h. We used Alexa Fluor 488 anti-mouse antibody (1:800, Jackson, Lancaster, PA, USA), Alexa Fluor 594 anti-rabbit antibody (1:800, Jackson), and Hoechst 33 342 (1:500, Thermo Scientific) as secondary antibodies. The stained samples were observed with a laser scanning confocal microscope (LSM800, Carl Zeiss AG) in selected channels (Alexa Fluor 594, Alexa Fluor 488, and Hoechst 33 342).
4
Protein Extraction and Western Blot Analysis
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Samples were homogenized using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA, or MCE, HY-K0010, Monmouth Junction, NJ, USA). After centrifugation at 13000 g for 15 min at 4°C, the supernatant of lysates was mixed with SDS-PAGE loading buffer (NCM Biotech, WB2001, Suzhou, China) and denatured at 100°C for 10 min for subsequent experiments. Western blotting (WB) analysis was performed as in our previous study (He et al., 2021 (link)) with primary antibodies: rabbit polyclonal anti-DNAH3 (Shanghai Youke Biotechnology Co. Ltd, customized, 1:300, Shanghai, China), rabbit polyclonal anti-DNAH2 (Novus Biologicals, NBP2-49506, 1:500, Littleton, CO, USA), rabbit polyclonal anti-DNAH6 (Abcam, ab122333, 1:500, Cambridge, MA, USA), rabbit polyclonal anti-DNALI1 (Abcam, HPA020046, 1:500), mouse monoclonal anti-GAPDH (Affinity Biosciences, T0004, 1:5000, Cincinnati, OH, USA), and mouse monoclonal anti-α-TUBULIN (Sigma, T8203, 1:5000 or Sigma, T6199, 1:3000, St Louis, MO, USA).
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