Fig. 5. Role of the cytoskeleton in Munc18-2 polarisation. RBL cells seeded on coverslips were treated with cytochalasin D (10 µM) or vehicles for 30 minutes (A) and nocodazole (33 µM) or vehicle for 1 hour (B) prior to incubation with antibodies to Munc18-2 (red fluorescence) and phalloidin-FITC (green fluorescence) or with antibodies to Munc18-2 (red) and -tubulin and RMCP II (both in green) as indicated. Cells were visualised by confocal microscopy as described in Materials and Methods. The merged images (green and red fluorescence) are shown, except for RMCP II staining, which is superimposed to the DIC image. Bars, 5 µm. Images are representative of at least three experiments. Note that the less focalised appearance of Munc18-2 staining in A is probably due to the solvent used for cell treatment. The nuclear appearance of Munc18-2 in nocodazole-treated cells in B (right panel) is caused by a low signal to noise ratio following redistribution of Munc18-2 to a diffuse staining pattern.