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Cyclic Multiplex Fluorescent Immunohistochemistry Protocol to Phenotype Glial Cells in Formalin-Fixed
Clara Muñoz-Castro1,2, Ayush Noori1,3, Bradley T Hyman1,2
1Massachusetts General Hospital Neurology Department, Boston, MA, USA.
Insights
This study introduces a cyclic multiplex fluorescent immunohistochemistry protocol for labeling up to 16 antigens on tissue sections. This method enhances cell phenotyping in health and disease research.
Area of Science:
- Biomedical research
- Cell biology
- Neuroscience
Background:
- Multiplexing immunohistochemistry is crucial for deep cell phenotyping in various biological contexts.
- Existing methods may have limitations in antigen labeling capacity and antibody compatibility.
Purpose of the Study:
- To describe a novel cyclic multiplex fluorescent immunohistochemistry protocol.
- To enable the detection of up to 16 antigens on a single tissue section.
- To facilitate deeper cell phenotyping in health and disease.
Main Methods:
- A cyclic multiplex fluorescent immunohistochemistry protocol was developed.
- The protocol utilizes "off-the-shelf" primary and fluorescently conjugated secondary antibodies.
- Key steps involve microwave-assisted antibody denaturation/stripping and signal quenching between cycles.
Main Results:
- Successfully labeled up to 16 antigens on formalin-fixed paraffin-embedded sections.
- Demonstrated the protocol's application in characterizing astrocytic and microglial responses in Alzheimer's disease brains.
- Validated the method for analyzing Aβ plaques and neurofibrillary tangles.
Conclusions:
- The described cyclic multiplex fluorescent immunohistochemistry protocol offers enhanced multiplexing capabilities.
- This adaptable method allows for detailed cell phenotyping across different tissues and diseases.
- It provides a valuable tool for advancing research in neuroscience and other fields.
Abstract:
There is a growing interest in expanding the multiplexing capability of immunohistochemistry to achieve a deeper phenotyping of various cell types in health and disease. Here, we describe a protocol of cyclic multiplex fluorescent immunohistochemistry that enables the labeling of up to 16 antigens on the same formalin-fixed paraffin-embedded section using "off-the-shelf," commercially available, primary antibodies as well as fluorescently conjugated secondary antibodies. Key steps include the denaturing/stripping of the antibodies by microwaving and the quenching of any remaining fluorescent signal between the cycles of otherwise traditional multiplexed fluorescent immunohistochemistry. We have successfully applied this protocol to characterize astrocytic and microglial responses to Aβ plaques and neurofibrillary tangles in Alzheimer's disease brains, but it could be easily adapted to other user's needs regarding cell types, disease, and organ.
