Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.
Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.
Takizawa
T
T
Department of Anatomy, Jichi Medical School, Tochigi, Japan. ttakizawa@jichi.ac.jp
Robinson
J M
JM
D005629:Frozen Sections; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009504:Neutrophils / Q000201:enzymology / Q000648:ultrastructure; D009195:Peroxidase / Q000378:metabolism; D010655:Phenylenediamines; D010879:Piperazines; D011435:Propyl Gallate
D007202:Indicators and Reagents; D010655:Phenylenediamines; D010879:Piperazines; D011435:Propyl Gallate; D009195:Peroxidase; C029728:4-phenylenediamine; C007306:triethylenediamine