Combined protein and DNA measurements by the ninhydrin-Schiff and Feulgen techniques

Abstract

Feulgen nuclear staining with pararosanilin-SO2 was combined with the ninhydrin-Schiff technique. The aldehyde groups converted from primary amino groups are stained with an acriflavine-Schiff reaction. This results in a red nuclear fluorescence and a bright yellow cytoplasmic and nuclear fluorescence. The combined fluorescence staining facilitates cytofluorometric determination of total protein and DNA in the same cell. The ninhydrin-Schiff reaction is affected by the fixation procedure and the duration of the ninhydrin reaction. Investigations with a model system showed that proportionality between the fluorescence intensity of acriflavine and the amount of protein stained by the procedure was obtained after fixation with a fixation mixture suggested by Böhm et al. (1968) and a reaction with ninhydrin at 37 degrees C for 10 h. The ninhydrin-Schiff reaction has no effect on the fluorescence intensity of cells previously treated with pararosanilin-Feulgen staining and it is not affected itself by this previous procedure. Testing this double fluorescence staining on cytology specimens taken from patients with gastric carcinoma and uterine cervial carcinoma, cancer cells were shown to have markedly increased protein and DNA contents compared with those of normal cells.

Applications of the Ninhydrin Reaction for Analysis of Amino Acids, Peptides, and Proteins to Agricultural and Biomedical Sciences

“…The discovery of the ninhydrin-Schiff staining technique stimulated widespread interest in applying the method to histochemistry. Examples include studies of (a) a ninhydrin-specific chemical test for hair keratin (219), (b) energy and amino acid metabolism in mouse brain (220), (c) protein and DNA content of cells (221), (d) the cytochemistry of hepatic lysosomes and their enzymatic aggregates by a ninhydrin-DMSO-thiosemicabazide silver protein reaction (222), and (d) the use of a ninhydrin reagent to assess histochemically protein-bound amino acids in rat tissues (223). The DMSO-H 2 O mixed solvent enhanced the rate of oxidative deamination and favored specific staining of tissues (222).…”

Determination of red cells, nucleic acid‐containing cells and platelets (RNP Determination) by a crossover analysis of emission DNA/RNA light

“…Supravital stains, such as new methylene blue, should be selected, and it is better that the dye penetrates cells easily. Acridine orange (Cambier, Wheeless & Patten, 1977;Fukuda et al, 1979Fukuda et al, , 1986Mezzanotte, Ferrucci & Marchi, 1981;, aminoacridines (Blake & Peacocke, 1968), acriflavine (Roth, 1976) or proflavine (Sagawa & Tatsumi, 1997) can be used to detect DNA by fluorescence. Acridine orange, acridine yellow or choriphosphine O are also used to detect RNA by metachromatic fluorescence (Bradley & Wolf, 1959).…”

Combined DNA and protein cytofluorometry of gynecological smears: Quantitative identification of tumor cells and discrimination of intraepithelial neoplasia from invasive cancer.

“…The combined DNA and protein staining was performed using the ninhydrinSchiff and Feulgen techniques as described previously (5,6,7). Briefly, the speci-mens were washed in running tap water for 2 hr and pre-warmed in distilled water at 60°C prior to hydrolysis.…”