Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections

Microsc Res Tech. 2005 May;67(1):15-21. doi: 10.1002/jemt.20181.


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.

MeSH terms

  • Antigens, CD / metabolism
  • Biomarkers / analysis
  • Biomarkers, Tumor / analysis*
  • Colorectal Neoplasms / immunology
  • Colorectal Neoplasms / pathology*
  • Fluorescent Antibody Technique
  • Humans
  • Immunohistochemistry*
  • Immunophenotyping*
  • Leukocytes / cytology*
  • Leukocytes / metabolism
  • Microscopy / methods*
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Palatine Tonsil / immunology
  • Palatine Tonsil / pathology


  • Antigens, CD
  • Biomarkers
  • Biomarkers, Tumor