Pneumologie 2019; 73(S 01)
DOI: 10.1055/s-0039-1678318
Freie Vorträge (FV DGP 13) – Sektion Allergologie und Immunologie
Pneumologische Immunologie und Allergologie
Georg Thieme Verlag KG Stuttgart · New York

Improved technique for in situ staining of murine and human pulmonary immune cells

W Martin
1   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, German Center for Infection Research (Dzif), Standort Hannover-Braunschweig, Experimental Virology and Twincore, A Joint Venture Between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany
,
R Grychtol
2   Department of Pediatrics, Allergology and Neonatology, German Center for Lung Research, Biomedical Research in End Stage and Obstructive Lung Disease/Breath Hannover; Pädiatrische Pneumologie, Allergologie und Neonatologie, Zentrum für Kinderheilkunde und Jugendmedizin, Medizinische Hochschule Hannover
,
AC Jirmo
3   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, Experimental Virology and Twincore, A Joint Venture Between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany
,
J Bergmann
4   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, German Center for Lung Research, Biomedical Research in End Stage and Obstructive Lung Disease/Breath Hannover, Germany
,
S Gläsener
5   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, Germany
,
P Braubach
6   German Center for Lung Research, Biomedical Research in End Stage and Obstructive Lung Disease/Breath Hannover, Department of Pathology, Hannover Medical School, Germany
,
C Dopfer
3   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, Experimental Virology and Twincore, A Joint Venture Between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany
,
A Habener
3   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, Experimental Virology and Twincore, A Joint Venture Between the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany
,
G Hansen
4   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, German Center for Lung Research, Biomedical Research in End Stage and Obstructive Lung Disease/Breath Hannover, Germany
,
C Happle
4   Department of Pediatrics, Allergology, and Neonatology, Hannover Medical School, German Center for Lung Research, Biomedical Research in End Stage and Obstructive Lung Disease/Breath Hannover, Germany
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Publikationsverlauf

Publikationsdatum:
19. Februar 2019 (online)

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    Purpose The analysis of the complex cellular pathologies underlying lung diseases is of growing scientific and diagnostic interest. However, to study the distribution and phenotype of specific lung cell subsets is a methodological challenge, requiring relatively large quantities of lung tissue. Here, we describe a novel technique for sustainable and in depth characterization of human and murine lung cells in situ.

    Material and Methods We optimized protocols for preparation, cryopreservation and analysis of human and murine lung tissue. After retrieval, specimens were prepared by incubation in sucrose and paraformaldehyde solutions and embedded into a molding compound. After freezing and cryotome-cutting, 3 µm thick slices were placed on a cytometric chip and, after staining of specific cell epitopes, subjected to iterative scanning on a fluorescence microscope. Systematic single cell analyses enabled photographic documentation and assessment of quantitative expression patterns.

    Results In both human and murine specimens, optimized cryopreservation and analysis techniques allowed for clear identification and localization of various specific immune cell subsets within the lung tissue. Using a mouse strain with spontaneous pulmonary inflammation due to lack of regulatory T-cells (Scurfy mice), we were able to demonstrate clear infiltrates of T helper and B cells in the lung tissue. In human lung specimens of a cystic fibrosis patient, we were able to show an increase of cytotoxic T cell infiltration and myeloid cell activation compared to the lungs of healthy donors. Preserved lung specimens were stable for several months, allowing for repeated staining of more than ten markers per cell.

    Conclusion The here described protocols were optimized for sustainable and in depth in situ characterization of immune cells in human and murine lung specimens. In contrast to previously described techniques, they permit for iterative staining of multiple markers to analyze phenotype and localization of specific cell subsets in small lung specimens, even after long time storage and with longer interruption between staining cycles. We believe the here described method may help to significantly improve scientific and diagnostic approaches in respiratory diseases.


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