Fig. 3. Localisation of Munc18 and syntaxins isoforms. (A) Co-localisation with PM marker. RBL cells were seeded overnight on glass coverslips. After permeabilisation, cells were incubated with antibody to FcRI ß chain (1 µg/ml) as a PM marker and either antibodies to Munc18-2, Munc18-3, syntaxin 3 or syntaxin 4 (all at 2 µg/ml). Cells were visualised by confocal microscopy as described in Materials and Methods. Single optical sections through individual cells as well as the merge (green and red fluorescence) from several cells are presented. In the inset of the upper panel the green and red fluorescence are superimposed to the DIC image. Arrows highlight area of red fluorescence in cellular protrusions. (B) Co-localisation with a SG marker. RBL cells were treated as above before incubation with antibody to RMCP II (1/500) as a SG marker, and antibodies to either Munc18-2 or syntaxin 3 (all at 2 µg/ml). For BMMCs, cells were allowed to adhere to L-polylysine-coated coverslips for 1 hour before staining with antibody to serotonin (1/50) as a SG marker, and antibodies to either Munc18-2 or syntaxin 3. Cells were visualised by confocal microscopy as described in Materials and Methods. Single optical sections through individual cells are presented. For BMMCs only the merge (green and red fluorescence) is shown. Bars, 5 µm. Images are representative of at least three experiments.