John W. Kelly

QUATERNARY AMMONIUM COMPOUNDS IN CONNECTIVE TISSUE HISTOCHEMISTRY: I. SELECTIVE UNBLOCKING'

Certain features of an accepted biochemical system for fractionating biological polyanions were adapted for more precise identification of acid mucopolysaccharides in histological preparations. Tissue studies were based on two major steps: (1) complete blocking of all polyanions by a quaternary ammonium detergent and (2) selective unblocking by increasing concentrations of an electrolyte. Critical stages were visualized by suitable stains for acid mucopolysaccharides. Results with tissues and certain polyanion models showed that both acid mucopolysaccharide and nucleic acid structures behaved histochemically, under proper conditions, as predicted from the biochemical reactions of such polyanions as hyaluronic acid, chondroitin sulfate, heparin, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Chemical fixation involving formaldehyde seriously altered the expected positions of nucleic acid structures in the unblocking sequence; this was corrected by use of cetylpyridinium chloride in the fixing solution. Considering the biochemical background of these methods and several different histochemical applications already accomplished, numerous possibilities are open for extending the fundamental and practical uses of quaternary ammonium compounds in microscopic studies.

A new fluorescent method with phenanthrenequinone for the histochemical demonstration of arginine residues in tissues.

Phenanthrenequinone, in an alkaline ethanolic solution, is shown to be specific for the guanidino group of arginine in tissues. The fluorescence measured in chick erythrocyte nuclei is highly reproducible among areas on one slide and among different slides. Benzil and cyclohexanedione, two agents known to be specific for the guanidino group, prevent 85% of the fluorescence developed in the phenanthrenequinone reaction. Glyoxal blocks phenanthrenequinone reactivity only partially, while alkaline formaldehyde has little effect. The effects of fixation and postfixation of chick erythrocyte nuclei indicate that fluorescence is most intense after formalin fixation, and that conformational factors may prevent ethanolacetic acid-fixed nuclei from reacting completely. By means of protein precipitation and prolonged incubation in ethanol, it was shown that the fluorescent chromophore is strongly bound to phenanthrenequinone-reacted gelatin and to tissue sections. Excitation and emission spectra are presented for phenanthrenequinone-reacted arginine, gelatin and a tissue section.

Paper Chromatography of Anionic Disazo Dyes, Especially Trypan Blue and Its Red Impurity

Twenty-one samples of 10 dyes were studied by paper chromatography, using Whatman #1 paper and developing by the ascending technique with 80% ethanol. Fifteen other Whatman papers were tested. Solvents faster than 80% ethanol (10% pyridine, 50% actone, 50% and 70% ethanol) were not useful. Slower solvents (isoamyl alcohol, isoamyl alcohol saturated with NH4OH, 90% acetone, acetone, 95% ethanol) were generally unsatisfactory but sometimes useful for extreme sharpness of spots produced in descending chromatograms. Blue and violet (reddish) fractions were found in trypan blue, Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B, and Niagara sky blue 6B. Trypan blue and Evans blue showed traces of additional fractions in acid or alkaline 80% ethanol or on acid-washed papers. Red, orange and yellow fractions were generally present in trypan red. A single red component was in Congo red and vital red and only a violet component in Congo corinth. A special study was made of one sample of trypan blu...

An evaluation of the metachromasy of anionic dyes. II. Visual and spectral observations on solutions.

The reactions of 13 anionic dyes in solution with a basic protein (protamine), a cationic detergent, guanidine, histamine, procaine, quinine, and strychnine were examined visually and spectrophotometrically in order to distinguish metachromatic changes of the dyes. Disazo dyes (Congo red, benzopurpurin, but not trypan blue) were metachromatic; indigoid, triphenylmethane and xanthene dyes were not. The magnitude of metachromasy in this series of dyes was not great compared with cationic dyes, the shifts of absorbance maxima being only about 15 mμ against 90 mμ or more for some cationic metachromatic dyes. The most effective chromotropes were protamine and a cationic detergent. Agreement between visual observations on tissue sections, visual observations on solutions, and spectral observations on solutions was generally good.

An evaluation of the metachromasy of anionic dyes. I. Visual observations on tissue sections.

Thirteen dyes of the azo (benzopurpurin, Congo red, trypan blue, chromotrope 2R, orange G), indigoid (indigocarmine), triphenylmethane (acid fuchsin, aniline blue, light green, methyl blue), and xanthene (eosin B, eosin Y, erythrosin B) groups were applied under standard conditions to a variety of human, rabbit, rat, mouse and frog tissues in paraffin sections. Sections were examined for color changes which might indicate metachromatic reactions analogous to the metachromasy of cationic dyes. Disazo and xanthene dyes showed shifts in hue, with some qualification on the shifts of xanthenes. Metachromatic shifts of anionic dyes were generally of low order compared to those of cationic dyes. Nuclei, erythrocytes, inner elastic laminae of arteries, keratinous structures, and certain areas in the ground substance of connective tissue most often elicited metachromasy. It is suggested that basic proteins are responsible for the metachromatic reactions. Equally interesting areas were those staining poorly (cartil...

THERMAL ANALYSIS OF POLYANION METACHROMASY: TEMPERATURE EFFECTS ON STAINED CELLS, TISSUES AND MODELS

Metachromasy of toluidine blue-stained materials was examined at 5-95°C visually and 30-70°C microspectrophotometrically. Cells and tissues provided sites of acid mucopolysaccharides and nucleic acids, which were also prepared as films and droplets. As in similar studies of aqueous solutions, metachromatic ratios were inverse, linear, reversible functions of temperature, with the possible exception of deoxyribonucleic acid. An aqueous mounting medium (gelatin) supported maximum excursions of metachromasy during heating and cooling, although reversible loss of metachromasy occurs to lesser degrees in conventional media. Removal or denaturation of cartilage matrix protein merely increased over-all metachromasy; slopes of thermal plots were unchanged. All evidence suggests that metachromasy is not a fundamentally different phenomenon in solutions and solid systems. Temperature studies emphasize the role of structured water in metachromasy, interaction of water and other solvents and particularly solvent dielectric constant in relation to dye-dye interaction. The limited literature on temperature and biologic staining is reviewed.

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.

PHOTOGRAPHIC CYTOPHOTOMETRY WITH A DUAL-MICROSCOPE. II. MICROSCOPE ASSEMBLY AND FILM-DYE EXTRACTIONS.

Improved apparatus for dual-microscope photographic cytophotometry is described. The major component is a new comparison microscope, available commercially, whose binocular system is optically corrected for performance equivalent to that expected of one microscope. While current applications involve only standard bright-field and interference optics for absorption and dry-mass studies, a feature of the assembly is ready interchangeability of optics for fluorescence, polarization, phase, and dark-field microscopy. A method for extraction of color-film dyes into dimethylsulfoxide is reported. The dye solutions are used for spectrophotometric measurement of amount of dye in a given area of film and, indirectly, for estimating the amount of chromophore in the corresponding area of a microscopic object.