Fig. 4. Munc18-2 localisation after FcRI-dependent stimulation and relation to cytoskeletal elements. (A) Analysis of the composition of a large Munc18-2-positive compartment. IgE-sensitised RBL cells seeded on coverslips were stimulated for 30 minutes with DNP-HSA and probed with antibody to Munc18-2 (2 µg/ml) for confocal analysis as described in Materials and Methods. Raw confocal acquisitions of a single cell were subjected to mathematical analysis using Huygens software to improve resolution. A DIC image of the cell as well as an expanded region containing a Munc18-2-positive compartment (red fluorescence), probably comprised of at least seven discernible SGs, is shown. Bar, 2 µm. (B) RBL cells were seeded on glass coverslips, sensitised with IgE overnight and either not stimulated (-Ag) or stimulated with DNP-HSA for 10 minutes (+Ag). Cells were processed for staining with antibodies to Munc18-2 (in red) and either phalloidin-FITC (1/40) or antibody to -tubulin (1 µg/ml) (both in green). Cells were visualised by confocal microscopy as described in Materials and Methods. Comparison of a single section, a 3D and a XZ projection are shown for stimulated cells. Single sections of unstimulated cells are shown in the inset. A DIC image of the cell stained for Munc18-2 and -tubulin is also shown as an inset. All size bars represent 5 µm.

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