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© 1996 Springer-Verlag. Reproduced with permission.
(from Durso et al. 1996, Protoplasma 190: 141-150)
a,b,c Dual immunolocalization and presumed FRET using confocal laser scanning microscopy on the same focal plane of a taxol-treated carrot protoplast's cortex. The image of the Texas Red fluorescence reporting anti-carrot tubulin mAb1F8 (a) is highly similar to the image of the fluorescein fluorescence reporting affinity-purified anti-pp50 antibodies (b). An image of the specimen excited for fluorescein, but observed for Texas Red (c) may be explained as a result of FRET in which excited fluorochromes associated with affinity-purified anti-pp50 transferred energy to excite anti-tubulin-associated fluorochromes.
d,e Control specimen: A single immunolocalization (using the same anti-tubulin method and the same excitation and observation parameters as a and c) and confocal laser scanning microscopy on the same focal plane of a taxol-treated carrot protoplast's cortex. Excitation and observation for tubulin (as in a) produces tubulin-associated fluorescence (d), whereas excitation and observation for FRET (as in c) results in no detectable fluorescence.
These data indicate that the fluorescence observed in c is the result of FRET; i.e., the laser line used to excite anti-pp50-associated fluorochrome does not directly excite the anti-tubulin-associated fluorochrome. FRET suggests that some pp50 is very closely associated (i.e., 50 Å) with microtubules upon fixation, in turn, indicating that the pp50/microtubule co-localization observed in footprints lysed prior to fixation is not the result of an artifactual redistribution of pp50. Bar = 4 um.
