Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion Export

@article{citeulike:4635955, abstract = {Abstract\ \ In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.}, author = {Mei{\ss}litzer-Ruppitsch, Claudia and Vetterlein, Monika and Stangl, Herbert and Maier, Susanne and Neum\"{u}ller, Josef and Freissmuth, Michael and Pavelka, Margit and Ellinger, Adolf}, citeulike-article-id = {4635955}, citeulike-linkout-0 = {http://dx.doi.org/10.1007/s00418-008-0429-4}, citeulike-linkout-1 = {http://www.springerlink.com/content/u75752230m924102}, day = {1}, doi = {10.1007/s00418-008-0429-4}, journal = {Histochemistry and Cell Biology}, keywords = {dab, gfp, grab, photoconvert}, month = {August}, number = {2}, pages = {407--419}, posted-at = {2009-05-26 13:29:31}, priority = {2}, title = {Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion}, url = {http://dx.doi.org/10.1007/s00418-008-0429-4}, volume = {130}, year = {2008} }

TY - JOUR ID - citeulike:4635955 L3 - citeulike-article-id:4635955 N2 - Abstract  In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research. IS - 2 JF - Histochemistry and Cell Biology EP - 419 TI - Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion VL - 130 SP - 407 KW - dab KW - gfp KW - grab KW - photoconvert AU - Meißlitzer-Ruppitsch, Claudia AU - Vetterlein, Monika AU - Stangl, Herbert AU - Maier, Susanne AU - Neumüller, Josef AU - Freissmuth, Michael AU - Pavelka, Margit AU - Ellinger, Adolf PY - 2008/08/01/ UR - http://dx.doi.org/10.1007/s00418-008-0429-4 ER -