FLUORESCENCE PHOTOOXIDATION WITH EOSIN - A METHOD FOR HIGH-RESOLUTIONIMMUNOLOCALIZATION AND IN-SITU HYBRIDIZATION DETECTION FOR LIGHT AND ELECTRON-MICROSCOPY

A simple method is described for high-resolution light and electron mi croscopic immunolocalization of proteins in cells and tissues by immun ofluorescence and subsequent photooxidation of diaminobenzidine tetrah ydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is ac hieved in the photooxidation process over other conventional fluoresce nt compounds. The technique allows for precise correlative immunolocal ization studies on the same sample using fluorescence, transmitted lig ht and electron microscopy. Furthermore, because eosin is smaller in s ize than other conventional markers, this method results in improved p enetration of labeling reagents compared to gold or enzyme based proce dures. The improved penetration allows for three-dimensional immunoloc alization using high voltage electron microscopy. Fluorescence photoox idation can also be used for high resolution light and electron micros copic localization of specific nucleic acid sequences by in situ hybri dization utilizing biotinylated probes followed by an eosin-streptavid in conjugate.