Computer analysis of double labeling: ultrastructural distribution of vasoactive intestinal peptide and transferrin...

Transferrin (Tf) and vasoactive intestinal peptide (VIP) were labeled with horseradish peroxidase (HRP) and 125I, respectively. To determine whether two simultaneously incubated ligands are conveyed by the same population of endosomal vesicles in human colon carcinoma cells (HT-29), we used an analysis system derived from the cross-fire method for quantitation of autoradiographic data. This system permitted the collection of data and the statistical calculations required by the double labeling of the cells. HRP-labeled Tf organelles were chosen as reference structures of the endosomal apparatus and taken as the conventional source of the radiolabeling. Our data established from the co-localization hypothesis strongly suggest that after a 30-sec (T 1/2) and a 10-min (T10) internalization at 37 degrees C, VIP and Tf share in major part the same endocytic pathway and even the recycling route to the cell surface. At T10, most of the radiolabeling was located inside the tubulovesicular network, and we also detected slight radiolabeling inside the vesicles recycling Tf. The number of double-labeled endosomes involved in ligand traffic were advantageously observed with our computer-assisted analysis system.