Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion.
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 gamma-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.
Meiblitzer-Ruppitsch
Claudia
C
Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University Vienna, Schwarzspanierstrasse 17, 1090, Vienna, Austria.
Vetterlein
Monika
M
Stangl
Herbert
H
Maier
Susanne
S
Neumüller
Josef
J
Freissmuth
Michael
M
Pavelka
Margit
M
Ellinger
Adolf
A
D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D001896:Boron Compounds / Q000032:analysis* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D055032:Electron Microscope Tomography / Q000379:methods*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005453:Fluorescence; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins / Q000032:analysis*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D051122:Scavenger Receptors, Class B / Q000378:metabolism; D033921:Vesicular Transport Proteins; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001426:Bacterial Proteins; D001896:Boron Compounds; D050485:GABA Plasma Membrane Transport Proteins; C060042:GOLGA5 protein, human; D000077614:Golgi Matrix Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C495124:SCARB1 protein, human; C493413:SLC6A1 protein, human; D051122:Scavenger Receptors, Class B; D033921:Vesicular Transport Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D015100:3,3'-Diaminobenzidine; D005680:gamma-Aminobutyric Acid; D019704:Protein Disulfide-Isomerases