Alexa fluor 594

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).

Two-photon microscopy of the peritoneal subserosa was performed as previously described.^{49 (link)} Mice were anesthetized with isoflurane, the parietal peritoneal wall was exposed through a midline incision, and sterile, pre-warmed HBSS buffer containing 0.2 μg anti-CD88 Alexa Fluor 594 (Biolegend; clone 20/70) was applied to the tissue surface after it had been mounted on a special base plate. After a 10-min rest period, 100 ng of LPS in HBSS was applied topically to the peritoneal serosa before a sterilized coverslip was placed on the surface. Immediately afterward, dynamic imaging was started on a Zeiss 710 microscope equipped with a femtosecond pulsed Chameleon laser (Coherent) and a 20× water immersion objective (NA 1.0, Zeiss). Data were acquired at a resolution of 512×512 (16 bits) in batches of 12 images, each 3 mm apart, at a frame rate of 968 ms using ZEN software (Zen 2012; Zeiss). For imaging, the laser was set to 930 nm with a power of approximately 80 mW and the following filter cubes were used: GFP (565DCXR, 525/50; Zeiss), Alexa Fluor 594 (565DCXR, 575–640; Zeiss), SHG (495DCLP, 447/60; Zeiss). Raw imaging data were processed and analyzed with Imaris (Bitplane).