Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea.
We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.
D002467:Cell Nucleus / Q000032:analysis; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007475:Ion Exchange Resins; D008297:Male; D008689:Methacrylates; D008856:Microscopy, Fluorescence; D008867:Microtomy; D008875:Middle Aged; D009925:Organ of Corti / Q000032:analysis* / Q000648:ultrastructure
D007475:Ion Exchange Resins; D008689:Methacrylates; C005044:hydroxyethyl methacrylate; D004247:DNA