Abstract
AbstractNecroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell deathin vivohas proven challenging in the absence of robust reagents and protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators – Caspase-8, RIPK1, RIPK3 and MLKL – in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells exhibited reduced RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors were observed in response to insults such as sterile inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. This toolbox and methods will facilitate precise localisation and evaluation of necroptotic signalingin vivo.
Publisher
Cold Spring Harbor Laboratory