Quantitative immunofluorescence analysis of microtubule concentration
and distribution in retracting axons.
After experimental manipulations, cells were fixed and prepared using a general antibody against -tubulin. Red represents the lowest fluorescence intensity and white represents the highest. a, Control axon. b, Axon treated with nocodazole for 30 min. c, d, Axons 30 min after injection of neurons with dynamitin. All axons except the control retracted by at least a third of their original lengths. Note that the nocodazole-treated axon shows a clear diminution in polymer concentrations, whereas axons of dynamitin-injected neurons show significantly higher concentrations of polymer. Total fluorescence intensities in a, c and d are roughly equal, showing that little or no microtubule depolymerization occurs during retraction. The high levels of fluorescence in distal regions of retracting axons indicate that microtubules may retreat disto-proximally from the axon tip during retraction. d shows that the trailing strands often left during dynamitin-induced retraction contain microtubules. Scale bar represents 18 Ám.