Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.
Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabelling in cultured cells
CISTERNA, BARBARA;PELLICCIARI, CARLO
2013-01-01
Abstract
Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
