Fluorescent porphyrin counterstaining of chromatin DNA in conjunction with immunofluorescence methods using...

This work describes the use of two cationic porphyrins for fluorescent DNA counterstaining of HeLa cells also stained by means of indirect immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Staining HeLa cells with meso-tetra(4-N-methylpyridyl)porphine (T4MPyP) and meso-tetra(p-N-trimethylanilinium)-porphine (TMAP), both used at 5 x 10(-6) M, gives rise to a deep red emission of chromatin from interphase nuclei and mitotic chromosomes when the cells are excited with blue (490 nm) light. The red-fluorescing chromatin contrasted very well with the yellowish-green emission from FITC-immunofluorescent staining. No significant difference in chromatin fluorescence found with either T4MPyP or TMAP was detected. Counterstaining with the porphyrins could be carried out as a separate step after immunolabeling or, more simply, by their inclusion in the mounting medium. Spectral analysis demonstrated that the fluorescent emission maximum of T4MPyP was at 665 nm and that the intensity of the fluorescent emission showed a considerable increase in the presence of DNA.