Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

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.