Fig. 2. Localization of Wnt proteins in the chick neural tube. Wnt1 and GFP (A-C) or Wnt3a and GFP (D-F) were expressed in the neural tube following electroporation. Diagram to left depicts GFP (green) expression in an electroporated neural tube; the boxed region is shown in A-F. GFP-positive embryos were harvested 24 hours post-electroporation (HH stage 18/19), fixed and cryosectioned prior to immunostaining with anti-Wnt1/Wnt3a antibodies. Goat anti-mouse-Cy3 secondary antibodies were used to visualize Wnt proteins (red). All images were collected by confocal microscopy. White arrows point to perinuclear staining; arrowheads indicate immunopositive punctae. No punctae were observed when primary antibody was omitted (data not shown). (G-O) A wild-type chick embryo (HH stage 18) was cryosectioned and stained with Wnt1 primary antibody and a Cy3-labeled secondary antibody (red). Diagram to left shows endogenous Wnt1 expression (red) in the neural tube; the boxed region is shown in G-O. G and H are adjacent sections, as are I-L. In H, primary antibody was omitted. In I, excess GST-Wnt1 blocking peptide was added. In J-L, we compare the localization of Wnt1 protein (J) with that of Wnt1 transcript (K). In M-O, we compare the localization of Wnt1 (red) with NCAM (green) in a single optical confocal layer. In M and O, white arrows point to staining on or just interior to the plasma membrane, whereas the white arrowheads indicate staining that is inside the cell. nt, neural tube; no, notochord.