Quantification by image analysis of immunocytochemical reactions: application for determination of lysozyme content in individual smeared cells.

The objective of the present study was to develop a cytophotometric technique to quantitate immunocytochemical reactions. Cell antigens were detected after immunophosphatase alkaline staining procedure. The amount of reaction product was quantitated by computerized scanning cytophotometry. The technical conditions (dilution of primary antibody; incubation time of the three antibodies; volume and pH of the enzyme substrate reaction; storage of the slides) required for optimal cytophotometric determination of the reaction product were determined. Under these optimally defined conditions, a linear relationship between cell protein content (lysozyme) and microdensitometric measure of the colored reaction product was found. This method could be used for other cells, antigens, and enzymatic indicators.

A cytophotometric method to quantitate the binding of monoclonal antibodies to individual cells.

A cytophotometric technique to quantitate binding of (monoclonal) antibodies to individual cells is described. This method is based on the detection of cell-surface antigens by immunoperoxidase staining procedures. The amount of reaction product of the peroxidase reaction with diaminobenzidine/H2O2 was quantitated by computerized scanning cytophotometry. The incubation period of peroxidase with the substrate required for an optimal cytophotometric determination of the reaction product proved to be 60 min. The reproducibility of individual absorbance measurements, the day-to-day reproducibility of the quantitation of monoclonal antibody binding, and the specificity and sensitivity of the detection of defined monoclonal antibodies, established the reliability of the present method. The sensitivity of the indirect immunoperoxidase procedure could be enhanced by using a biotin-avidin-peroxidase staining procedure.

Analysis of the changes in the morphology and staining density characteristics of cartilagenous cell nuclei from Triturus cristatus during aging and regeneration.

The modifications in the morphology and staining intensity of cell nuclei reflect their physiologic and functional state. In order to appreciate the variations in the metabolic activity of hind limb chondrocytes from Triturus cristatus during aging and regeneration, we have studied the nuclear size and shape, the DNA concentration, and the distribution and degree of condensation of the chromatin. This study was carried out using scanning cytophotometry on semithin 1 micrometer sections, Feulgen-stained for DNA, which is the major nuclear component. During aging, the nuclei decrease in size, becoming irregular in shape. The concentration of DNA increases and the chromatin distribution becomes more heterogeneous with an increase in condensed chromatin and a subsequent decrease in the diffuse chromatin fraction. During regeneration, however, these modifications are reversed whatever the age of the animal, but even more noticeably when the animal is old. The changes that occur in the chromatin fractions defined according to their state of condensation are discussed from a functional point of view.

A reliable Feulgen-acriflavine-SO2 staining procedure for quantitative DNA measurements.

Feulgen-acriflavine-SO2 staining was performed on chicken erythrocytes and rat liver cells under different cytochemical conditions. The obtained results were compared to conventional Feulgen-pararosaniline-SO2 staining by means of scanning cytophotometry and microfluorometry. It was found that if acid ethanol rinsing was included in the Feulgen-acriflavine-SO2 procedure, both Feulgen procedures were fully comparable in specificity and quantitative aspects.