Tcs sp8 confocal mp flim

Spermatozoa collected from cauda epididymides were resuspended in prewarmed (37°C) phosphate-buffered saline (PBS) containing 200 nM MT-DR FM and incubated at 37°C for 45 min. After staining, the sperm were pelleted again by centrifugation and resuspended in prewarmed PBS. This spermatozoon suspension was spotted on glass slides and air-dried. The nuclei of cells on the slides were counterstained with Hoechst 33342 and mounted with antifade mounting medium. All samples were observed by laser scanning confocal microscopy (TCS SP8-Confocal-MP-FLIM, Leica, Mannheim, Germany).

Spermatozoa were collected by density gradient centrifugation, using Percoll solution (Sigma, St. Louis, USA) as a medium [23 ]. All Percoll solutions were buffered with Biggers, Whitten, and Whittingham medium (BWW). Following centrifugation, sperm that accumulated at the bottom of the tube were collected.
According to the operation manual recommended by Life Technologies Corporation, spermatozoa were resuspended in prewarmed (37°C) BWW containing 200 nM MitoTracker Deep Red FM (MT-DR FM) (Invitrogen, Carlsbad, USA) and incubated at 37°C for 45 min. After staining, the spermatozoa were washed, spotted on glass slides, and air-dried. These preparations were fixed with 4% paraformaldehyde and then permeabilized with ice-cold acetone. After blocking with 10% goat serum, spermatozoa were sequentially incubated with anti-DJ-1 monoclonal antibody (1 : 200, Abcam, Cambridge, UK) or anti-NDUFS3 monoclonal antibody (1 : 200, Abcam, Cambridge, UK) and then goat anti-rat lgG secondary antibody (1 : 200, Alexa Fluor 555 conjugate) or goat anti-mouse lgG secondary antibody (1 : 200, Alexa Fluor 488 conjugate) (Thermo Fisher Scientific Inc., Rockford, USA). Normal rabbit or mouse IgG was used as a negative control. Nuclei were counterstained with Hoechst 33342. All samples were observed by laser scanning confocal microscopy (TCS SP8-Confocal-MP-FLIM, Leica, Mannheim, Germany).

The sections were viewed under confocal microscope (TCS SP8‐Confocal‐MP‐FLIM; Leica) with 561 nm Argon/Krypton laser line exciting the Alexa Fluor. MEP images were collected with a 10× microscope objective. Once a fluorescent signal was positive in the microscopic field, 60‐μm thick stacks in Z‐axis (2 μm steps) were acquired, as the area of interest was confirmed, with a 40× oil immersion microscope objective. Each optical section was then added to a stack of images using Leica data processing software (LAS AF, Leica, Heidelberg, Germany).