Figure 4

Live GFP-mouse mammary gland compared with the identical whole mounted gland. GFP-mouse mammary gland from a 4.5 week old mouse (gland number 3) was prepared as described in Methods and imaged using a Zeiss LD PAPO 25x/0.8 lens (Table 1). A. SHG-B/SHG-F were imaged in A and SHG-B/ BP 500-550/SHG-F were imaged in B. Orthogonal views are compared. In the live tissue XZ view (on top left), blue represents the unfiltered ChD transmitted signal which includes SHG-F (white arrow) and the GFP fluorescence (blue of epithelial cells, dotted arrow). In whole mount tissue (at right), the SHG-F (blue, white arrow) is more intense. The signal from unfiltered ChD also includes the Carmine Alum fluorescence (remaining blue associated with epithelial cells). The XY view illustrates SHG-B (red) of the surface fibrillar layer (Z = 1 and Z = 6, respectively, for live and whole mount). B. Orthogonal views made at different Z-depths illustrate increased information provided by combination of SHG signals (SHG-B, red and ChD unfiltered, including both SHG-F plus GFP in the live and SHG plus Carmine Alum fluorescence in the whole mount). Arrows indicate points of comparison between live and whole mount tissue. Asterisks indicate a fiber-associated vessel. C. The TEB shown in A-B is included in this lower magnification image taken using a Zeiss Neofluar 10x/0.30 lens (asterisk, green GFP, red, SHG-B). D. A kymograph was generated from a line with average 100 pixel width. The line was similarly placed on the image of the live and whole mount TEB and the image merged. SHG-B from the whole mount appears in red, and the SHG-B from the live appears in green. A-B, Scale bar = 50 μm, C, Scale bar = 100 μm.