A reliable and sensitive method for fluorescent photoconversion.
The objective of this study was to develop a reliable and sensitive method of diaminobenzidine (DAB) photoconversion through systematic variation of the objectives and the method of DAB enhancement employed. Results showed that reliable photoconversion of DiI-labeled macaque monkey cortical cells and processes can be obtained consistently by using fresh filtered DAB solutions, clean slides, and a bright fluorescent light source. Using a 20x objective and enhancing the DAB reaction product with Giemsa stain provided the most sensitivity and best resolution in the photoconverted area. Objective size was found to correlate directly with sensitivity and indirectly with both the size of the area photoconverted and the time to complete photoconversion. Nitro blue tetrazolium (NBT) did not produce as much contrast in the photoconverted cells as Giemsa stain, but allowed for the best visualization of finer processes because it somewhat enlarged the fiber diameter. Nickel and cobalt used together, but not separately, were found to provide adequate enhancement. These results are likely to apply to photoconversion and enhancement of all fluorescent material, but may require slight modifications in protocol.
Singleton
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Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2175, USA.
Casagrande
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D015100:3,3'-Diaminobenzidine; D000818:Animals; D001399:Azure Stains; D002232:Carbocyanines; D005453:Fluorescence; D005456:Fluorescent Dyes; D008029:Lighting; D008254:Macaca nemestrina; D009488:Neurosciences / Q000379:methods*; D009580:Nitroblue Tetrazolium; D012680:Sensitivity and Specificity; D014793:Visual Cortex / Q000166:cytology / Q000648:ultrastructure
D001399:Azure Stains; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009580:Nitroblue Tetrazolium; D015100:3,3'-Diaminobenzidine; C024286:3,3'-dioctadecylindocarbocyanine