A double-labeling technique usign fluorescent compounds, i.e., diamidino yellow and true blue, is described, with the divided rat sciatic nerve model. Tibial and peroneal fascicles were transected, and each freshly cut nerve end was placed in a vial containing either the true blue or the diamidino yellow label. After a 10-15 day survival period, animals were systematically perfused with fixative, and the lumbar enlargement and dorsal root ganglia corresponding to sciatic nerve roots were removed. Serial sectioning and examination with ultraviolet fluorescence microscopy and counts of the labeled cells were performed. Diamidino yellow in labeled cells was found localized to the nucleus, whereas true blue was found localized to the cytoplasm and nucleus (control group n = 5). Intermixing of these pools was noted, with yellow cells seen among blue labeled cells following nerve autograft repair of a 1 cm segment of the rat sciatic nerve (experimental group n = 5). Double-labeled cells displaying a yellow nucleus and a blue cytoplasmic label were also seen. We believe that the double-labeling technique described, using the fluorescent retrograde tracer diamidino yellow and true blue, is a useful monitoring method for assessing the specificity of regenerating axons in the rat sciatic nerve.
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