Three-dimensional colocalization analysis in immunostained sections of human pancreas

Abstract

We describe here how to perform colocalization analysis in different endocrine cells of human pancreatic tissue. Immunostained endocrine cells were imaged using high resolution three-dimensional (3D) confocal microscopy. The analysis of the 3D images was performed using Volocity software based on the fluorescence intensity of the different markers acquired in separate channels. The described procedure can be used for colocalization analysis in immunostained tissues other than pancreatic islets. Special attention should be paid to the limits of the imaging resolution and quality for proper colocalization analyses, especially, those involving sub-cellular compartments. Execution of the described procedure will highly depend on the computer processing speed; in our hands, it took a few minutes per islet. However, the overall time of analysis will vary significantly depending on the sampling size to accomplish statistical power of analysis.