Prediction of in situ fluorescence of histochemical reagents using a structure-staining correlation procedure

A total of ninety acid, basic, and non-ionic dyes were screened for fluorescent staining of various Carnoy fixed rat tissues. It was found that the fluorescence/non-fluorescence of a dye could be predicted using a conjugated bond number (CBN) cut-off value. Thus 90% of dyes with CBNs of 29 or less were fluorescent; whilst 70% of dyes whose CBNs exceeded 30 were nonfluorescent. The cut-off value was not significantly influenced by the charge, or the hydrophobic-hydrophilic character of the dye; though fluorescence was greatly influenced by the mode of fixation. The CBN cut-off value proved surprisingly robust. Thus most fluorochromes found in the histochemical literature have small conjugated systems, with CBNs less than the cut-off value. This includes labels of immunoglobulins, vital stains of neurones, and fluorescent Schiff reagents. Conversely several dyes used to quench background autofluorescence have large conjugated systems, with CBNs substantially above the cut-off value.