Single-laser three-color immunolabeling of a histological section by laser scanning microscopy: application to senile...

We describe a method for observing three-color immunofluorescence simultaneously in a histological section under a confocal laser scanning microscope. In this study we investigated the spatial relationship of blood vessels, reactive microglia, and amyloid beta-protein (A beta) deposits in post-mortem brain tissue of patients with Alzheimer's disease. HLA-DR was employed as a marker for activated microglia and von Willebrand factor (vWF) as a marker for vascular endothelial cells. HLA-DR was labeled with R-phycoerythrin (R-PE) and vWF with fluorescein isothiocyanate. A beta-protein was immunostained with the tandem conjugate of R-PE and cyanin 5. Three images were obtained serially by scanning a tissue section with a 488-nm laser beam in combination with an appropriate emission filter for each fluorochrome. Overlaying of these three images permitted simultaneous observation of three distinct structures: activated microglia, blood vessels, and A beta deposits. This technique provides an improved way to study the localization of three different antigens in a single tissue section.