Anti ddx4 antibody

E. invadens trophozoites and cysts were fixed with acetone/methanol (1:1) and permeabilized with 0.1% Triton X-100. Cells were incubated with 3% bovine serum albumin (BSA) for blocking followed by mouse monoclonal anti-NF-YA antibody (Santa Cruz; catalog no. sc-17753) (1:200) or anti-NF-YC antibody (Abcam; catalog no. ab232909) from rabbit (1:500) followed by Alexa Fluor 488 anti-mouse or anti-rabbit antibody, respectively (Molecular Probes) (1:2,500). Localization was performed with anti-DDX4 antibody (Thermo Fisher) from mouse (1:200), followed by Alexa Fluor 543-conjugated anti-mouse secondary antibody (Molecular Probes) (1:2,500). Slides were prepared using Vectashield mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Inc.) and visualized using a Leica CTR6000 microscope and a BD CARVII confocal unit. Images were analyzed using Leica LAS-AF software.

As a biological control, DEAD box polypeptide 4 (Ddx4) (Fujiwara et al., 1994 (link); Castrillon et al., 2000 (link)), an evolutionarily conserved germ cell-specific RNA helicase, was detected in oocytes from the IVG system. Three oocytes were assessed in both groups. Germinal vesicle (GV) oocytes that failed to mature after oocyte IVM were fixed in 4% paraformaldehyde (Servicebio, Wuhan, China) for 30 min, and then transferred to drops of 0.5% TRITON X-100 (ZSGB-BIO, Beijing, China) for 30 min. Oocytes were blocked in 3% BSA diluted in phosphate-buffered saline (PBS; pH 7.4, Gibco). One hour later, oocytes were incubated with anti-DDX4 antibody (1:200; ABCAM; Cambridge, UK) overnight at 4 °C followed by incubation with Alexa Fluor 647 goat anti-mouse IgG (1:500; Thermo Fisher, Carlsbad, CA, USA) for 1 h. Cell nuclei were stained with DAPI dihydrochloride (Invitrogen). Confocal images were acquired using a Leica Corp. TCS SP8 confocal system (Leica Corp. Microsystems, Heidelberg, Germany).