An immunohistochemical atlas of necroptotic pathway expression

Author:

Chiou Shené,Al-Ani Aysha H.,Pan Yi,Patel Komal M.,Kong Isabel Y.,Whitehead Lachlan W.ORCID,Light Amanda,Young Samuel N.,Barrios Marilou,Sargeant Callum,Rajasekhar Pradeep,Zhu Leah,Hempel Anne,Lin Ann,Rickard James A.,Hall Cathrine,Gangatirkar Pradnya,Yip Raymond K.H.,Cawthorne Wayne,Jacobsen Annette V.,Horne Christopher R.,Ioannidis Lisa J.,Hansen Diana S.,Day Jessica,Wicks Ian P.,Law Charity,Ritchie Matthew E.,Bowden Rory,Hildebrand Joanne M.,O’Reilly Lorraine A.,Silke John,Giulino-Roth Lisa,Tsui Ellen,Rogers Kelly L.ORCID,Hawkins Edwin D.,Christensen Britt,Murphy James M.ORCID,Samson André L.ORCID

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

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