Assessment of Lung Eosinophils In Situ Using Immunohistological Staining
Christopher D Nazaroff1,2, William E LeSuer1, Mia Y Masuda1
1Division of Allergy, Asthma, and Clinical Immunology, Department of Medicine, Mayo Clinic Arizona, Scottsdale, AZ, USA.
Abstract:
Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.
Insights
Detecting eosinophils in allergic lung inflammation is challenging. This study presents antibody staining methods for precise identification of eosinophils and their granule proteins in mouse lung tissue, improving diagnostic accuracy.
Area of Science:
- Immunology
- Cell Biology
- Respiratory Medicine
Background:
- Eosinophils are key inflammatory cells in allergic respiratory diseases.
- Traditional hematoxylin-eosin (HE) staining often makes eosinophil detection difficult in lung tissue.
- Degranulation and cell lysis further complicate eosinophil identification using HE staining.
Purpose of the Study:
- To develop and validate antibody-based methods for specific eosinophil detection.
- To enable clear visualization of eosinophils and their granule proteins in mouse lung.
- To overcome limitations of HE staining for identifying eosinophils in allergic inflammation models.
Main Methods:
- Utilized eosinophil-specific anti-mouse antibodies for detection.
- Applied antibody staining to formalin-fixed, paraffin-embedded lung tissue (in situ).
- Performed antibody staining on cytospin preparations from lung cells.
Main Results:
- Successfully detected eosinophil granule proteins using antibody staining.
- Enabled both colorimetric and fluorescence imaging of eosinophils.
- Demonstrated the ability to detect degranulated or lysed eosinophil remnants.
Conclusions:
- Antibody staining provides superior specificity and clarity for eosinophil identification compared to HE.
- These methods are valuable for studying allergic respiratory inflammation in mouse models.
- The techniques allow for multiplexing with additional antibodies for comprehensive analysis.
