Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Lavinia Schoenfeld; Birgit Appl; Laia Pagerols-Raluy; Annika Heuer; Konrad Reinshagen; Michael Boettcher

doi:10.3791/65272

Journal

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Immunology and Infection

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Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

5,834 Views

•

07:36 min

•

August 18, 2023

DOI:

10.3791/65272-v

DOI:

10.3791/65272-v

•

07:36 min

August 18, 2023

•

87 Views

Lavinia Schoenfeld¹, Birgit Appl¹, Laia Pagerols-Raluy¹, Annika Heuer³, Konrad Reinshagen¹, Michael Boettcher^1,2

¹Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf,University of Hamburg, ²Department of Pediatric Surgery, University Medical Center Mannheim,University of Heidelberg, ³Division of Spine Surgery, Department of Trauma and Orthopedic Surgery,University Medical Center Hamburg-Eppendorf (UKE)

Transcript

Among antibody selection for tissue imaging, the fixation process poses a challenge by epitope masking. Therefore, customized retriever step is required to expose these antigens for antibody binding. Additionally, tissue inherent autofluorescence can cause difficulties through to a high background staining.

However, the use of different approaches in the protocols in the literature hinders a standardized comparison between the images. Our study aims to address the demand for standardized procedures and assist the researcher in overcoming challenges during the imaging process. We established a versatile imaging protocol applicable to different tissues by comparing various different protocols.

So our work provides guiding and troubleshooting tips from antibody usage to sample mounting, highlighting the potential pitfalls. Our findings advance research by introducing a new primary antibody for citrullinated hisone three, and an autofluorescent reducing agent to minimize background staining. Furthermore, for successful imaging, we emphasize the careful handling of samples and the importance of maintaining a constant high temperature for antigen retrieval.

Summary

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.