Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections. 
Immune-cell infiltration is frequently seen within human solid tumors.  A detailed phenotypic analysis of these cells may aid in the understanding of an antitumor immune response.  Standard hematoxylin/eosin and conventional immunohistochemical stainings are helpful, but have major limitations in the number of markers that can be identified and localized per tissue section.  Therefore, we developed a combined fluorescence and brightfield microscopic technique by using both immunofluorescence and immunogold-silver methods, thereby discriminating three different leukocyte markers plus one tumor marker simultaneously in a single section.  This enabled us to study both phenotype and location of infiltrating immune cells in colorectal tumors.  We used a two-step staining in which primary and secondary antibodies were selected for minimal cross-reactivity.  Furthermore, the secondary fluorescent antibody conjugates were selected for minimal spectral overlap.  For computer-assisted analysis the brightfield microscopy image was combined with the fluorescence microscopy images.  This combination of techniques provides a powerful tool for detailed multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissue sections in general and for tumor-immune cell infiltration in particular.