Differential distribution of acetylated and tyrosinated MTs in hippocampal neurons. (A–H) Polarized stage 3 (A–D) and morphologically unpolarized stage 2 (E–H) rat hippocampal neurons stained for acetylated (B and F) and tyrosinated (C and G) α-tubulin (arrows, axons; arrowheads, minor neurites). Cells were permeabilized during fixation to remove unpolymerized tubulin subunits, therefore only tubulin incorporated in MTs was assessed. In stage 3 neurons, a high ratio of acetylated to tyrosinated α-tubulin is found in MTs in the axonal shaft (D, arrow) in comparison to MTs of minor neurites (D, arrowheads, and I). In 35.0 ± 6.1% of morphologically unpolarized stage 2 neurons, the ratio of acetylated/tyrosinated α-tubulin is significantly increased in one of the minor neurites (H, white arrowhead with asterisk; P < 0.05 by Hampel outlier test). The areas boxed in H are shown in higher magnification in K and M. (I and J) Ratio quantification of fluorescence intensities of acetylated and tyrosinated α-tubulin in MTs of stage 2 (J) and 3 (I) neurons (mean ± SEM; n > 105 neurons from three independent experiments for each stage 2 and 3). Values are normalized to the mean of nonaxonal or nonmaximal processes for stage 3 and 2, respectively. ***, P < 0.001 by t test. (K–N) Higher magnification views (K and M) and profiles of immunofluorescence intensity (in arbitrary units; L and N) of acetylated and tyrosinated α-tubulin of the neurites marked in H. Bars: (A–H) 20 μm; (K and M) 10 μm.