Combined immunocytochemical staining and image analysis for the study of lymphocyte specificity and function in situ

Immunocytochemical staining methods were combined with computer-aided image analysis to quantitate relative intracytoplasmic and cell membrane products within single lymphoid cells. Lymphoid cells fixed to microscope slides were immunocytochemically stained and the densities of immunocytochemical stains for individual lymphoid cells were determined on video-camera-captured microscopic images to quantitate synthesized and fully assembled antibody molecules detected by an anti-idiotypic antibody. Similarly, expression of a lymphoid cell membrane molecule, Ly-1, was comparatively quantitated in individual lymphoid cells. Densitometric analysis of tissue sections was utilized to measure tissue areas and quantitate cell nuclei in tissues. Two-color immunocytochemical staining and sequential image analyzer-determined locations of individual cells in tissues allowed unambiguous identification of doubly stained cells in their native tissue environment. This analysis was applied to the in situ identification of antibody forming cells producing the CRIc idiotype of the BALB/c anti-arsonate response and a determination of the mu chain usage by those individual cells. Applications of combined immunocytochemistry and computerized image analysis to studies of in vivo immunologic functions are discussed.