Nbp2 16109

Zebrafish embryos at different stages of development were fixed overnight in cold 4% paraformaldehyde-PBS (PFA). Embryos were then dehydrated with methanol for storage at −20°C and rehydrated, washed in PBST [PBS+0.1% Tween 20], permeabilized with cold acetone (20 s), blocked with goat serum for at least 1 h at room temperature. Thereafter, they were incubated with rabbit anti-P54 serum (1:2000), rabbit pre-immune serum, rabbit anti-Dcp2 (Novus Biologicals, NBP2-16109; 1:2000), rabbit anti-TIAL-1 (Novus Biologicals ,NBP1-79932; 1:2000), rabbit anti-phosphorylated non-muscle myosin (NMII-p) (Cell Signaling, 3671) or rabbit anti-DDX4 (anti-Vasa) (Abcam, ab13840), these were diluted in PBST and incubated overnight at 4°C. Labeling was detected using secondary antibody (1:1000) goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Jackson ImmunoResearch, 111-545-003), and nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole).

For whole-mount IF of embryos or ovaries, tissue was fixed in 4% paraformaldehyde overnight at 4°C, dehydrated in MeOH, and placed at—20°C. Two anti-Rbpms2 antibodies were used in this study, mαRbpms2 (Abcam, ab169394) a mouse polyclonal raised to full-length human protein (amino acids 1–209 NP_919248), and rαRbpms2 (abcam, ab170777) a rabbit polyclonal antibody raised to a peptide within amino acids 120–149 were diluted at 1:500, Anti-Bucky ball y1165 at 1:500 [14 (link)], and anti-GFP antibody (Invitrogen, A10262) was used at 1:500. Chicken anti-Vasa antibody was a gift of Bruce W. Draper and used at 1:1000 dilution. Rabbit anti-ACF7/Macf1 was used at 1:1000 [52 (link)]. Rabbit anti-DCP2 (Novus Biologicals, NBP2-16109) and rabbit anti- TIAL-1 (Novus Biologicals, NBP1-79932) were used at1:2000 as in [29 (link)]. Alexafluor488, Alexafluor568, CY3, C5 (Molecular Probes) secondary antibodies were diluted at 1:500. Images were acquired using a Zeiss Axio Observer inverted microscope equipped with Apotome or ApotomeII and a CCD camera, or Zeiss Live DuoScan (line-scanning) Confocal. Images were processed in ImageJ/FIJI, Adobe Photoshop and Adobe Illustrator.