Live duoscan

In zebrafish, Vasa protein localizes in a perinuclear ring of staining around each PGC nucleus that can be used much like a nuclear marker to identify and count individual cells. Z-series image stacks of one lateral half of embryonic gonads were obtained using a Zeiss Live DuoScan (line-scanning) confocal microscope, and cells in the stack were manually counted by analyzing the slices and nuclear morphology comprising each Z-stack with ImageJ/FIJI.

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.