Cy5-NP41 (acetyl-SHSNTQTLAKAPEHTGC-(Cy5)-amide) labeling of sciatic nerve in Thy1-YFP transgenic mice. (a) Low-power brightfield view of left exposed sciatic nerve. Inset shows magnified view of central boxed region. (b) Same nerve as in a with YFP fluorescence (pseudocolored yellow) superimposed on the brightfield image, showing transgenic expression of YFP in axons. (c) Same nerve as in a and b viewed with Cy5 fluorescence (pseudocolored cyan for maximal contrast during live surgery) superimposed on the brightfield image, showing nerve labeling with Cy5-NP41. Arrows in b and c point to thin buried nerve branches that are better revealed by the long-wavelength Cy5 fluorescence than by brightfield reflectance or shorter-wavelength YFP fluorescence. There is some nonspecific labeling of skin (asterisk) and cut edges of muscle (arrowhead) by Cy5-NP41. Fortunately, such nonspecific labeling hardly ever has the filamentous appearance of nerves, so an experienced surgeon can usually distinguish nonspecific from specific targets. (d) Low magnification longitudinal section showing myelin within the sciatic nerve using differential interference contrast (DIC), pseudocolored blue. (e) Same nerve as in d showing axoplasmic YFP pseudocolored green (arrows). (f) Same nerve as in d and e, showing Cy5-NP41 labeling (pseudocolored red) of epineurium (arrows) and endoneurium (arrowheads). (g) Composite image of d, e and f showing that NP41 labeling does not colocalize with either myelin or axoplasm. (h–k) Cross-sectional images corresponding to panels e–g.