Fig. 7. Secretory granules. Analysis for colocalization between CgA fragment-GFP chimeric photoproteins and dopamine ß-hydroxylase (DßH), a dense-core secretory (chromaffin) granule marker. (A) 48 hours after transfection with expression plasmids encoding SgP-GFP (a), SgP-CgA_1-77-GFP (b), SgP-CgA_1-115-GFP (c), SgP-CgA_1-224-GFP (d), SgP-CgA-GFP (e), SgP-CgAC₁₇E-GFP (f), SgP-CgA_233-439-GFP (g) or proANF-GFP (h), cells were aldehyde-fixed and processed for immunocytochemistry. Cells were incubated with a mouse anti-rat DßH followed by Alexa-Fluor-594 (red)-conjugated goat anti-mouse. GFP was excited at _ex 490±10 nm and imaged at _em 528±38 nm; the red Alexa Fluor 594 conjugate was excited at _ex 555±28 nm and imaged at _em 580±20 nm. Nuclei were visualized with Hoechst 33342 (_ex 350 nm/_em 461 nm). Optical sections along the Z axis were acquired with increments of 0.2 µm with a Delta Vision imaging system using a 100x oil immersion objective. Volume views (left panel) of the GFP chimeras and Alexa-Fluor-594-conjugated antibody distributions are shown. The extent of intracellular 3D colocalization of red (Alexa 594) and green (GFP) fluorescent signals was assessed using Nearcount Image Analyzer software. Representative 0.2 µm XY optical sections acquired in the middle region of the cells, and corresponding three-color mask images generated by Nearcount Image Analyzer are shown. (B) Nearcount image quantification of GFP (green) chimeras and DßH (red) 3D colocalization. The area of analysis (crop region) was defined as a rectangle that included the whole cell. `Overlap' is defined as the total number of red (Alexa 594) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel at the same location (same pixel). `Nearness' is defined as the total number of red (Alexa 594) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel within a 3D 2x2x2 pixels window centered on a green (GFP) pixel (i.e. ±2 pixels). At the total magnification used here, a chromaffin granule spans 9-15 pixels (as determined by CgA-GFP or proANF-GFP). Areas of overlap or nearness are displayed in yellow (`mask'). Bar, 5 µm.

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