Related Experiment Video
Updated: Aug 7, 2026
Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions
Published on: September 23, 2013
Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and
Ronald L P van Vlierberghe1, Maro H Sandel, Frans A Prins
1Department of Surgery, Leiden University Medical Centre, Leiden, the Netherlands.
Insights
Researchers developed a novel microscopy technique to simultaneously identify multiple immune cell markers and tumor markers in a single tissue section, enhancing the study of tumor-infiltrating immune cells.
Area of Science:
- Oncology
- Immunology
- Microscopy
Background:
- Immune-cell infiltration is common in human solid tumors, crucial for understanding antitumor responses.
- Conventional staining methods (hematoxylin/eosin, immunohistochemistry) have limitations in multiplexing markers per section.
- Detailed analysis of immune cell phenotype and location within tumors is challenging.
Purpose of the Study:
- To develop an advanced microscopy technique for simultaneous multiparameter analysis of tumor-infiltrating immune cells.
- To overcome the limitations of conventional staining methods in identifying multiple markers.
- To enable detailed study of immune cell phenotype and location in colorectal tumors.
Main Methods:
- Developed a combined immunofluorescence and immunogold-silver staining technique.
- Utilized a two-step staining protocol with antibodies selected for minimal cross-reactivity.
- Selected secondary fluorescent antibody conjugates for minimal spectral overlap.
- Integrated brightfield and fluorescence microscopy images for computer-assisted analysis.
Main Results:
- Successfully discriminated three leukocyte markers and one tumor marker simultaneously in a single tissue section.
- Enabled detailed analysis of both phenotype and location of infiltrating immune cells.
- Demonstrated a powerful tool for multiparameter microscopic analysis of formalin-fixed, paraffin-embedded tissues.
Conclusions:
- The developed combined microscopy technique offers a significant advancement for studying tumor-immune cell infiltration.
- This method allows for more comprehensive analysis of immune cell populations within solid tumors.
- It provides a powerful tool for detailed multiparameter microscopic analysis in various tissue types.
Abstract:
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.
