Abstract
Confocal immunofluorescence microscopy is an advanced imaging technique routinely applied in the laboratory and clinics. Histological analyses are performed from tissue material. In general, a single fluorochrome per laser is employed, limiting simultaneous analysis to four antigens in one staining with a conventional 4-laser line microscope. Here, we describe a protocol for combining fluorochromes with the same excitation but different emission properties that allows for the analysis of six different antigens in confocal immunofluorescence microscopy with a conventional 4-laser line microscope. The proposed multiplexed method permits the identification and characterization of complex cell populations in rare tissue material.
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Acknowledgments
We thank S. Beck for technical support. We are grateful for the support by the Medical Immunology Campus Erlangen (MICE) and the Optical Imaging Center Erlangen (OICE). This work was partly supported by grants from the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG)] to D.D. (CRC1181-TPA7), D.D., and A.-S.S. (RTG1962). D.D. and A.-S.S. are funded by the Emerging Fields Initiative BIG-THERA of the Friedrich-Alexander University Erlangen-Nürnberg. L.H. was supported by Erlanger Leistungsbezogene Anschub-finanzierung und Nachwuchsförderung (ELAN) (DE-17-09-15-1-Heger). D.D. received support from Interdisziplinäres Zentrum für Klinische Forschung (IZKF) (IZKF-A65).
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Heger, L., Lühr, J.J., Amon, L., Smith, AS., Eissing, N., Dudziak, D. (2021). Six-Color Confocal Immunofluorescence Microscopy with 4-Laser Lines. In: Zamir, E. (eds) Multiplexed Imaging. Methods in Molecular Biology, vol 2350. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1593-5_2
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DOI: https://doi.org/10.1007/978-1-0716-1593-5_2
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