Human pancreas sections were deparaffinized, followed by acidic-pH heat–mediated antigen retrieval. Cultures of single pancreatic islet cells were fixed with 4% paraformaldehyde. Samples were blocked and permeabilized in PBS with 0.3% Triton X-100 and 10% goat or donkey serum. Primary antibodies CHOP (1:100; sc-575, Santa Cruz Biotechnology), GAD6 mouse monoclonal antibody (mAb) against C-terminus of GAD65 (31) (1:1,000); N-GAD65 mouse mAb against the N-terminus of GAD65 (32) (1:300), giantin (1:1,000; ab24586, Abcam); insulin (1:10,000; 4011-01, Linco); insulin (1:2,000; ab14042, Abcam), and CD3 (1:30; M7254, Dako) were incubated overnight at 4°C in PBS with 0.3% Triton X-100 and 1% goat or donkey serum. Alexa Fluor conjugated secondary antibodies (Molecular Probes) were incubated at 1:200 dilution in PBS with 0.3% Triton X-100 for 30 min at room temperature.Image Capture, Analysis, and QuantificationSamples were imaged on a Zeiss LSM700 confocal microscope with 63×/1.40 NA Plan-Apochromat oil-immersion objective for single islet cells and 40×/1.30 NA Plan-Apochromat oil-immersion objective for pancreatic tissue sections. All images for quantification within a single experiment were captured with the same laser power and detector gain. The ratio of GAD65 mean fluorescence intensity (MFI) in the Golgi compartment and post-Golgi vesicles compared with the rest of the cytosol was calculated with a custom ImageJ macro. Individual β-cells in a given field of view were identified and outlined by hand. For each cell, the macro automatically defined a region of interest (ROI) outlining the Golgi compartment, identified by giantin costain or by characteristic morphology and brightness thresholding of GAD65 stain, and GAD65+ vesicles, identified by brightness thresholding of GAD65+ bright puncta. A second ROI defined the remainder of the cell, excluding the Golgi, GAD65+ vesicles, and nucleus. GAD65 Golgi accumulation was reported as the ratio of MFI for the two ROIs.