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Combining immunodetection with histochemical techniques: the effect of heat-induced antigen retrieval on picro-Sirius
1Centre for Liver Disease Research, School of Medicine, The University of Queensland, Translational Research Institute, Brisbane, Australia (VLG) v.gadd@uq.edu.au.
Insights
This study presents a method for combining Sirius red staining with multi-label immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This approach allows for simultaneous visualization of collagen fibers and multiple antigens, improving fibrosis research.
Area of Science:
- Histology
- Immunohistochemistry
- Pathology
Background:
- Picro-Sirius red stains collagen fibers (fibrosis) in tissues.
- Multi-label immunohistochemistry visualizes cell types, locations, and antigen co-localization.
- Combining these methods offers insights into fibrosis in chronic diseases.
Purpose of the Study:
- To develop a method for simultaneous Sirius red staining and multi-label immunohistochemistry.
- To overcome challenges in combining these techniques on FFPE tissues.
- To enable visualization of antigens in relation to fibrosis.
Main Methods:
- Utilized formalin-fixed, paraffin-embedded (FFPE) tissue specimens.
- Investigated antigen retrieval methods compatible with Sirius red counterstaining, focusing on reduced heat and enzymatic digestion.
- Developed a detailed protocol for simultaneous antigen and Sirius red detection.
Main Results:
- Identified challenges in combining standard antigen retrieval with Sirius red staining due to tissue modification and collagen shrinkage.
- Demonstrated that reduced heat and enzymatic digestion are compatible antigen retrieval methods.
- Provided a validated method for simultaneous detection.
Conclusions:
- Simultaneous detection of antigens and Sirius red in FFPE tissues is achievable.
- Optimized antigen retrieval methods are crucial for preserving collagen integrity.
- This combined technique enhances the study of fibrosis and associated cellular changes.
Abstract:
Picro-Sirius red is a routine diagnostic stain intended for the histological visualization of collagen fibers (fibrosis) in tissue. Multi-label immunohistochemistry is a powerful tool used by researchers to visualize different cell types and their location within a tissue specimen, and to observe co-localization of antigens. Combining the specificity of immunodetection with the simplicity of Sirius red staining will allow researchers to visualize multi-antigen detection in relation to fibrosis, a common histological feature of injury in many chronic diseases. Pre-treatment of formalin-fixed, paraffin-embedded tissue (FFPE) specimens with antigen retrieval is essential for the work-up of most commercially available antibodies. The most common form of antigen retrieval involves boiling tissue specimens in buffer to break the cross-linkages caused by formalin fixation. However, this method causes tissue modification and collagen fiber shrinkage leading to suboptimal results when counterstaining for Sirius red. Reduced heat and enzymatic digestion are antigen retrieval methods compatible with Sirius red counterstaining. This paper will discuss the difficulties faced when combining these two staining methods, and provide a detailed method for the simultaneous detection of antigen and Sirius red in FFPE tissues.
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