Semiquantitative study of the distribution of two cellular antigens by computer-directed color analysis

The general way to differentiate objects in a field is to stain each of them specifically with a different dye, as the eye can easily discriminate colors. The use of specific dyes or immunohistochemical staining associated with an automated image analysis facilitates the quantitation of differently Colored objects.

Rabbit aortic smooth muscle cells were seeded in culture conditions. When the cells reach confluence, they can be stained by immunocytochemistry. We were able to compare four different variables of immunostaining of two antigens in the same preparation. Antigenic expression was detected after double immunocytochemical staining, alkaline phosphatase, or peroxidase, and each antigen was stained sequentially by either enzyme.

We have developed an approach to a nondestructive and noncolored limited quantitative method, that allows a strict analysis on the same view of two defined colors. We used the system of intensity, hue, and saturation to digitize colored video image. The distribution of these two antigens, alpha-actin and 2P1A2 both specific of smooth muscle cell, was quantitatively analyzed, and was not affected by the sequence of staining.

This technique opens new possibilities for pathologists to compare antigenic distribution after immunocytochemical labeling. This can be extended to other dyes on histologic sections and to facilitate diagnosis.