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Immunofluorescence Microscopy for DIGE-Based Proteomics
Rustam R Mundegar1, Margit Zweyer1, Dieter Swandulla2
1Institut für Physiologie II, Rheinische Friedrich-Wilhelms-Universität Bonn, Nußallee 11, 53115, Bonn, Germany.
Insights
Fluorescence immunohistochemistry verifies protein alterations in tissues at the cellular level. This method allows for visual detection of changes in protein abundance, such as dystrophin in skeletal muscle.
Area of Science:
- Biochemistry
- Cell Biology
- Histology
Background:
- Proteome alterations in tissues can be validated using immunohistochemistry.
- Fluorescence immunohistochemistry utilizes fluorescently labeled antibodies for protein detection.
- This technique allows for cellular-level analysis of protein expression.
Purpose of the Study:
- To outline a protocol for fluorescence immunohistochemistry.
- To demonstrate the detection of dystrophin in skeletal muscle sections.
- To provide a method for verifying proteomic changes at the cellular level.
Main Methods:
- Sample preparation and tissue sectioning.
- Immunostaining with primary and secondary antibodies.
- Fluorescent microscopy for target antigen detection.
Main Results:
- Visual comparison of fluorescent signals indicates significant alterations in protein abundance.
- Fluorescence immunohistochemistry provides cellular-level expression data.
- The protocol is applicable for detecting specific proteins like dystrophin.
Conclusions:
- Fluorescence immunohistochemistry is a valuable tool for validating proteomic findings.
- It offers a cellular-level perspective distinct from large-scale analyses.
- The described protocol facilitates dystrophin detection in skeletal muscle.
Abstract:
Alterations in the proteome of a tissue in different settings, as assessed by difference gel electrophoresis, can be verified for single proteins using immunohistochemistry. In fluorescence immunohistochemistry, an antibody to a particular antigen is applied to tissue sections, and fluorophores conjugated to a secondary antibody allow for the detection of target antigen with fluorescent microscopy. Visual comparison is sufficient for the detection of significant alterations in the abundance of a certain protein in different settings. Additionally, unlike large-scale proteome analyses and Western blot methods, expression of target protein can be analyzed at the cellular level by immunohistochemistry. In this chapter, a protocol for the application of fluorescence immunohistochemistry for the detection of dystrophin in skeletal muscle sections is outlined, including sample preparation, tissue sectioning, and immunostaining.
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