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.
van Vlierberghe
Ronald L P
RL
Department of Surgery, Leiden University Medical Centre, Leiden, the Netherlands.
Sandel
Maro H
MH
Prins
Frans A
FA
van Iersel
Liselot B J
LB
van de Velde
Cornelis J H
CJ
Tollenaar
Rob A E M
RA
Kuppen
Peter J K
PJ
D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis*; D015179:Colorectal Neoplasms / Q000276:immunology / Q000473:pathology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D014066:Palatine Tonsil / Q000276:immunology / Q000473:pathology
D015703:Antigens, CD; D015415:Biomarkers; D014408:Biomarkers, Tumor