Single-laser three-color immunolabeling of a histological section by laser scanning microscopy: application to senile plaque-related structures in post-mortem human brain tissue.
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.
Uchihara
T
T
Department of Neurology, Faculty of Medicine, Tokyo Medical University, Japan.
Kondo
H
H
Akiyama
H
H
Ikeda
K
K
D000544:Alzheimer Disease / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D016229:Amyloid beta-Peptides / Q000032:analysis*; D001344:Autopsy; D001921:Brain / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007834:Lasers; D017628:Microglia / Q000378:metabolism; D018613:Microscopy, Confocal; D014841:von Willebrand Factor / Q000032:analysis*
D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D006684:HLA-DR Antigens; D014841:von Willebrand Factor