Confocal fluorescence and immunohistochemistry. The whole coding sequence of mouse Trs4 cDNA was cloned in-frame into pEGFP-N1 to construct a Trs4-GFP recombinant plasmid. The Trs4-GFP-fusion protein was expressed in HeLa
AZ 960 using Lipofectamine 2000 transfection reagent. Anti-Trs4 antibody (1:500) and TRITC-conjugated anti-rabbit secondary antibody (1:200) were used for indirect immunostaining. To investigate the localization of Trs4 and Ddc8 proteins in mature spermatozoa, mouse cauda epididymidis was isolated and incubated in DMEM medium. The dissociated sperm were collected and smeared on glass slides. After fixing and blocking, the slides were incubated with anti-Trs4 or anti-Ddc8
antibodies (1:400) or preimmune rabbit serum (negative control). Subsequently, the slides were incubated with FITC-conjugated anti-rabbit secondary antibody (1:200). The nuclei were counterstained with 4′,6-diamidino-2-phenylindol (DAPI) and the images were captured using a laser confocal microscope (Zeiss). Immunohistochemistry was performed as described [11].