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Imaging Keratan Sulfate in Ocular Tissue Sections by Immunofluorescence Microscopy and LA-ICP-MS
Burak Boyraz1,2, Jessica Saatz3, Inga-Marie Pompös4
1Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Augustenburger Platz 1, Berlin 13353, Germany.
Abstract:
Carbohydrate-specific antibodies can serve as valuable tools to monitor alterations in the extracellular matrix resulting from pathologies. Here, the keratan sulfate-specific monoclonal antibody MZ15 was characterized in more detail by immunofluorescence microscopy as well as laser ablation ICP-MS using tissue cryosections and paraffin-embedded samples. Pretreatment with keratanase II prevented staining of samples and therefore demonstrated efficient enzymatic keratan sulfate degradation. Random fluorescent labeling and site-directed introduction of a metal cage into MZ15 were successful and allowed for a highly sensitive detection of the keratan sulfate landscape in the corneal stroma from rats and human tissue.
Insights
Monoclonal antibody MZ15 effectively detects keratan sulfate, a key component of the extracellular matrix. This antibody, when labeled, offers a sensitive method for studying corneal stroma changes in disease.
Area of Science:
- Biochemistry
- Immunology
- Histology
Background:
- Extracellular matrix (ECM) alterations are hallmarks of various pathologies.
- Carbohydrate-specific antibodies are crucial for monitoring ECM changes.
- Keratan sulfate is a significant glycosaminoglycan in the ECM, particularly in the cornea.
Purpose of the Study:
- To characterize the keratan sulfate-specific monoclonal antibody MZ15.
- To evaluate MZ15's utility in detecting keratan sulfate in tissue samples.
- To establish sensitive detection methods for keratan sulfate in the corneal stroma.
Main Methods:
- Immunofluorescence microscopy on tissue cryosections and paraffin-embedded samples.
- Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS).
- Enzymatic degradation using keratanase II.
- Random fluorescent labeling and site-directed metal cage introduction into MZ15.
Main Results:
- The keratan sulfate-specific monoclonal antibody MZ15 was thoroughly characterized.
- Enzymatic pretreatment with keratanase II confirmed efficient keratan sulfate degradation, preventing antibody staining.
- Successful labeling of MZ15 enabled highly sensitive detection of the keratan sulfate landscape.
- MZ15 detected keratan sulfate in the corneal stroma of both rat and human tissues.
Conclusions:
- The monoclonal antibody MZ15 is a valuable tool for detecting keratan sulfate.
- Labeled MZ15 provides a sensitive method for visualizing the keratan sulfate distribution in the corneal stroma.
- This approach aids in understanding ECM alterations in pathological conditions affecting the cornea.
