An immunohistochemical atlas of necroptotic pathway expression
Shene Chiou, Aysha Al‐Ani, Yi Pan, Komal Patel, Isabella Y. Kong, Lachlan Whitehead, Amanda Light, Samuel N. Young, Marilou Barrios, Callum J. Sargeant, Pradeep Rajasekhar, Leah Zhu, Anne Hempel, Ann Lin, James Rickard, Cathrine Hall, Pradnya Gangatirkar, Raymond K. H. Yip, Wayne Cawthorne, Annette V. Jacobsen, Christopher R. Horne, Katherine Martin, Lisa J. Ioannidis, Diana S. Hansen, Jessica Day, Ian P. Wicks, Charity W. Law, Matthew E. Ritchie, Rory Bowden, Joanne M. Hildebrand, Lorraine A. O’Reilly, John Silke, Lisa Giulino‐Roth, Ellen Tsui, Kelly L. Rogers, Edwin D. Hawkins, Britt Christensen, James M. Murphy, André L. Samson
Abstract
Necroptosis 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 death in vivo has proven challenging in the absence of robust 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 lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo.