FLUORESCENT PORPHYRIN COUNTERSTAINING OF CHROMATIN DNA IN CONJUNCTIONWITH IMMUNOFLUORESCENCE METHODS USING FITC-LABELED ANTIBODIES

This work describes the use of two cationic porphyrins for fluorescent DNA counterstaining of HeLa cells also stained by means of indirect i mmunofluorescence with fluorescein isothiocyanate (FITC)-conjugated se condary antibodies. Staining HeLa cells with meso-tetra(4-N-methylpyri dyl)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 o f chromatin from interphase nuclei and mitotic chromosomes when the ce lls are excited with blue (490 nm) light. The red-fluorescing chromati n contrasted very well with the yellowish-green emission from FITC-imm unofluorescent staining. No significant difference in chromatin fluore scence found with either T4MPyP or TMAP was detected. Counterstaining with the porphyrins could be carried out as a separate step after immu nolabelling 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 emi ssion showed a considerable increase in the presence of DNA