The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.
[Background] Characterisation of neutrophil distribution within the skin following inflammation represents an important avenue for the understanding of their specialized functions. Even though the recruitment of neutrophils to the inflamed skin has been widely characterized by flow cytometry (Hampton et al., 2015; Stock et al., 2014), such technique provides limited insight on the intravascular versus interstitial localization of neutrophils. Recirculating and tissue-localized neutrophils exhibit different phenotypes and functions, which necessitates their discrimination to identify key players of the local immune response. With the development of intravital microscopy (IVM), the direct visualization of fluorescently tagged immune cells in vivo is made possible. However, due to the high cost of IVM, the accessibility of this powerful imaging tool to researchers is limited. Here, we present an alternative protocol for the high resolution static imaging of neutrophils in skin by making use of cost-effective reagents and commonly available confocal laser-scanning microscopy.
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