Alexa fluor 647 anti mouse igg

The PCAGGS-HSP90A-His and pcDNA3.1-KNG1-3 × Flag or pcDNA3.1-MAVS-Flag plasmids were transfected into HepG2.2.15 cells, which were grown on coverslips for 48 h. HepG2.2.15 cells were washed three times with PBS before fixation with 4% formaldehyde for 20 min. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 min and then blocked with 5% bovine serum albumin for 1 h. Cells were incubated with the corresponding antibodies for 1 h at room temperature. After washing three times with TBST, cells were incubated for 1 h at room temperature with Alexa Fluor® 488 conjugated anti-rabbit IgG (#ab150077, Abcam) and Alexa Fluor® 647 anti-mouse IgG (#ab150115, Abcam). DAPI (#P0131, Beyotime) was used for nucleus staining. The intracellular localization was analyzed by a confocal microscope (Nikon, Tokyo, Japan).

Sperm were collected from the cauda of the epididymis with a 27-G syringe in HTF medium. Additionally, the sperm cells localized in seminiferous tubule were collected from testis using 0.1% collagenase contained HTF medium. After incubation for 30 min and centrifuging, the pellet was washed using 5% (w/v) BSA/PBS. The sperm or sperm cells were fixed with 100% methanol for 10 min at room temperature and then permeabilized by 0.1% (v/v) TritonX-100/PBS for 10 min. Sperm were incubated in 5% (w/v) BSA/PBS for 30 and then probed with the primary antibody (anti-TLR7 antibody conjugated Alexa fluor 647 [bs-6601R-A647,Bioss], anti-TLR8 antibody, or anti-acetylated tubulin antibody [#T7451; Sigma] were used at 1:100). After washing by 5% (w/v) BSA/PBS, the antigens were visualized with Alexa fluor 488 anti-rabbit IgG (1:200; # 4030–30, Southern Biotech, Birmingham, AL, USA), Alexa fluor 647 anti-mouse IgG (1:200; #ab150115, Abcam), or DAPI (VECTESHIELD Mounting Medium with DAPI, Vector Laboratories, Burlingame, CA, USA). The intensity was analyzed by flow cytometry (Attune NxT Acoustic Focusing Cytometer, Invitrogen).