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Updated: Aug 9, 2026
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Constant detection of CD2, CD3, CD4, and CD5 in fixed and paraffin-embedded tissue using the peroxidase-mediated
1Department of Pathology, Medical University of Lübeck, Lübeck, Germany.
Insights
A new ImmunoMax method enhances immunohistochemical detection of leukocyte antigens in formalin-fixed tissues. This technique optimizes fixation, unmasking, and antibody binding for improved diagnostic accuracy in histopathology.
Area of Science:
- Histopathology
- Immunohistochemistry
- Molecular Biology
Background:
- Immunohistochemistry (IHC) is crucial for histopathology diagnostics.
- Monoclonal anti-leukocyte antibodies typically require frozen tissues.
- Formalin fixation, while optimal for morphology, can mask or destroy antigen binding sites.
Purpose of the Study:
- To develop a method for detecting formalin-sensitive leukocyte antigens in routinely processed tissues.
- To overcome the limitations of antigen masking in formalin-fixed, paraffin-embedded (FFPE) tissues.
- To enhance the sensitivity of IHC for leukocyte markers.
Main Methods:
- Developed the ImmunoMax method, optimizing tissue fixation and antigen retrieval.
- Employed enhanced immunohistochemical techniques to increase sensitivity.
- Utilized conventional monoclonal antibodies on FFPE lymphoid tissue specimens.
Main Results:
- The ImmunoMax method significantly enhances IHC sensitivity for leukocyte antigens.
- Successfully detected CD2, CD3, CD4, and CD5 antigens in FFPE tissues.
- Demonstrated that leukocyte antigens are masked, not irreversibly destroyed, by formalin fixation.
Conclusions:
- The ImmunoMax method enables reliable detection of formalin-sensitive leukocyte antigens in FFPE tissues.
- Optimized pretreatment and enhanced IHC sensitivity are key to retrieving masked antigens.
- This technique expands the utility of IHC in histopathological diagnosis using standard tissue processing.
Abstract:
Immunohistochemical methods are widely used for diagnostic purposes in histopathology. However, the use of most monoclonal anti-leukocyte antibodies is limited to frozen tissues. Initially, it was believed that formalin fixation in particular, which is the gold standard for morphological tissue preservation, destroys most of the antigen binding sites. In recent years, protease digestion and the introduction of microwave techniques have significantly enhanced the sensitivity of immunohistochemical techniques, and a variety of hidden antigen sites in formalin-fixed tissue have been retrieved for initially unreactive antibodies. It therefore became clear that many of the leukocyte antigens are not irreversibly destroyed but are most probably masked during the fixation process. We developed a technique combining optimized pretreatment of formalin-fixed tissue with a dramatic enhancement of the immunohistochemical sensitivity and named it the ImmunoMax method. The ImmunoMax method proves that by optimizing the technique at the following three levels it is possible to detect formalin-sensitive leukocyte antigens: (a) standard fixation of the tissue; (b) sufficient antigen unmasking; and (c) increasing the substrate turnover by multiplication of binding sites with subsequent enhancement of the immunohistochemical reaction. Using this optimized ImmunoMax method, we were able to detect CD2, CD3, CD4, and CD5 with conventional monoclonal antibodies in formalin-fixed, paraffin-embedded tissue specimens of various lymphoid tissues.
