Electron-dense markers

The fluorescent labels reported for investigation of intracellular uptake and distribution by CLSM comprise Nile red [13], Texas Red, and 6-coumarin [14]. Not only for fluorescence microscopy but also for transmission electron microscopy (TEM), the loading of markers proved to be useful. Osmium tetroxid as an electron dense marker and bovine serum albumin (BSA) as a model protein were entrapped in PLGA-nanoparticles to elucidate their uptake and intracellular distribution in human vascular smooth muscle cells [15]. 

In applying this system, a biotinylated binding molecule (e.g., antigen, primary or secondary antibody) is allowed to interact with a target system (cells, microtiter plates, and so on). By uang an appropriate avidin-conju-gated probe (e.g., fluorescent or electron-dense marker, solid support, enzyme), the system can be used for a variety of different applications. 

The specimen is prepared to expose antigens at the surface of the sample, and these samples are successively incubated with primary antibody, a wash, and a secondary antibody that is complexed with an electron dense marker. Blocking steps are included to reduce nonspecific attachment of the reagents to either the sample or the background. 

Reisfeld et al. (1987) described a hydrazide biotin labeling method (Table 7.20D) by which they could detect 1 pg of target DNA. Hydrazide compounds can be used similarly for a large number of other labels (enzymes, chromo- or fluorophores, electron-dense markers, etc.). Since the reaction is specific for ss nucleic acid, loops in duplices can be specifically labeled. 

With the introduction of cytochemical labelling methods a new field in electron microscopy was opened up and has allowed the localization of specific molecules, such as proteins and glycolipids, with a high resolution and specificity. These methods are based upon a wide variety of marker molecules, such as fluorescent markers for light microscopy, introduced by Coons [131] and electron dense markers for electron microscopy, which were introduced by Singer [132],... 

---