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).
Hpa017382
Manufactured by Merck Group
Sourced in Australia
HPA017382 is a laboratory equipment product manufactured by Merck Group. It is designed for use in scientific research and analysis applications. The core function of this product is to facilitate controlled and precise measurements or manipulations in a laboratory setting. No further details on the intended use or specific applications of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Hpa038440, by Merck Group (3 mentions)
Hpa021624, by Merck Group (3 mentions)
Nbp2 14260, by Novus Biologicals (3 mentions)
Ab122333, by Abcam (2 mentions)
Ab122367, by Abcam (2 mentions)
Hpa020046, by Merck Group (2 mentions)
T6793, by Merck Group (2 mentions)
Hpa040343, by Merck Group (2 mentions)
Bx51 fluorescence microscope, by Olympus (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)
Sds loading buffer, by Beyotime (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
Alexa fluor 488, by Yeasen (1 mentions)
Alexa fluor 594 antibodies, by Jackson ImmunoResearch (1 mentions)
4 protocols using hpa017382
1
Immunofluorescence Analysis of Sperm Ultrastructure
2
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).
3
Proteomic Analysis of Human Spermatozoa
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Human spermatozoa from AY078 and control fertile groups were washed three times with PBS, dissolved using 1×SDS loading buffer (Beyotime Biotechnology,China), and denatured at 100°C to avoid protein loss due to inadequate lysis. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:1000), rabbit polyclonal anti-SPAG6 (HPA038440, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:1000), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:1000), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:1000), and mouse polyclonal anti-β-actin (TA-09, ZSGB-Bio, China). After incubation with secondary antibodies at 37°C for 2 h, blots were visualized and analyzed(Tanon 5200,China).
4
Immunofluorescence Analysis of Sperm Structure
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IF was performed after samples had been pre-processed as described previously (32 (link)) using rabbit polyclonal anti-HYDIN (HPA067155, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-SPEF2 (HPA040343, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-PLCζ1 (pab0367-P, Covalab, USA, 1:100), rabbit polyclonal anti-ACTL7A (HPA021624, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-ACROSIN (NBP2-14260, Novus Biologicals, Colorado, USA, 1:200), rabbit polyclonal anti-RSPH1 (HPA017382, Sigma, Castle Hill, NSW, Australia, 1:100), rabbit polyclonal anti-RSPH3 (17603-1-AP, Proteintech, Rosemont, IL, USA, 1:100), as well as mouse monoclonal anti-acetylated α-tubulin (T6793, Sigma, Castle Hill, NSW, Australia,1:500) antibodies and secondary anti-mouse Alexa Fluor 488 (Yeasen Biotechnology, USA, 34106ES60, 1:500) and anti-rabbit Alexa Fluor 594 antibodies (Jackson ImmunoResearch, USA, 111–585-003, 1:500). DNA was stained using Hoechst 33342 (Thermo Fisher Scientific, USA, 62,249, 1:1000).
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