Localization of microtubule plus-ends in proplatelets. (A-B) Megakaryocytes extending proplatelets were permeabilized with Triton X-100 and incubated with TRITC (tetramethylrhodamine-isothiocyanate)–tubulin as described in “Materials and methods.” (A) Fluorescence micrograph showing a permeabilized proplatelet after incubation with rhodamine-tubulin for 4 minutes. TRITC-tubulin incorporates into specific foci along the length of the proplatelet. Bar, 4 μm. (B) Differential-interference-contrast micrograph of cell labeled in panel A. (C-D) Anti-EB3 immunofluorescent labeling of a proplatelet-containing megakaryocyte. EB3 staining (arrowheads label cometlike dashes) is dispersed along proplatelets and is abundant in the cell bodies (CB) but is not found in a radial pattern (compare with preproplatelet megakaryocytes in Figure 5). The boxed region in panel C shows a high-magnification view of the comets. Bars, 4 μm. (C, inset) The immunoblot shows that anti-EB3 antibodies recognize a 36-kDa polypeptide in both (L1) preproplatelet megakaryocytes and (L2) proplatelet-containing megakaryocytes. MW lane indicates molecular weight protein ladder.