Photoconversion of fluorescent retrograde tracers

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

“…In contrast to Bentivoglio and Su (1990), we did not find that imaging the area to be photooxidized before the addition of DAB seriously interfered with subsequent photooxiclarion, as long as photobleaching was kept to a minimum. Antifade media were not used during imaging because we found that they may interfere with subsequent photooxidation in the imaged area.…”

“…This phenomenon may account for the observation of Bentivoglio and Su (1990) that photooxidation can take place even when there is no fluorescence. Fixation with glutaraldehyde and treatment with 0.06 M potassium cyanide following immunolabeling but before photooxidation greatly reduced mitochondrial staining.…”

“…The reaction product is highly insoluble and easily visible with ordinary transmitted light microscopy. Since the DAB reaction product is osmiophilic, this technique can be used for correlative light and electron microscopic studies using a variety of fluorescent compounds including Lucifer yellow, DiI, Bodipy ceramide, fluorogold, and fluororuby (Pagano et al, 1989;Bentivoglio and Su, 1990;yon Bartheld et al, 1990;Balercia et al, 1992;Lubke, 1993;Papadopoulos and Dori, 1993;Schmued and Snavely, 1993;Takizawa et al, 1993). A potentially powerful application of this technique is for correlated light and electron microscopic studies of immunolabeled specimens.…”

Current concepts in neuroanatomical tracing

“…Longer survival times increase¯uorescence, but then dye leakage occurs, resulting in staining of surrounding glial cells and a decrease in the number of labelled neurones. In this respect other dyes with similar applications [FG, Mini Ruby (MR), Fluoro-Ruby (FR), Fluoro-Emerald (FE)] are even worse than FB (Akintunde and Buxton, 1992;Bentivoglio and Su, Bentivoglio et al, 1980;Novikova et al, 1997;Vince et al, 1997).…”

Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section

“…Because of its excellent and stable fluorescence, simplicity of application, easy reproducibility of the results and many other unique properties, this fluorescent dye has become widely used for tracing neuronal connections (Pieribone and Aston-Jones, 1988;Schmued and Heimer, 1990;Divac and Mogensen, 1990;Wessendorf, 1991;Akintunde and Buxton, 1992;Schmued, 1994;Novikova et al, 1997). Drawbacks with respect to the demands for multiple tracing listed above is that this dye belongs to the family of fluorescent tracers, and that FG is not electrondense in itself and therefore is not very useful in ultrastructural analysis without the help of labor-and time-consuming post-staining processing like photoconversion (Bentivoglio and Su, 1990;Balercia et al, 1992). In this regard, the introduction of sensitive antisera against FG has allowed 'change of family' and opened the possibility of using a permanent chromogen in the light microscopical detection of the dye (Chang et al, 1990;Van Bockstaele et al, 1994).…”