An immunohistochemical atlas of necroptotic pathway expression

Chiou, S.; Al-Ani, A.; Pan, Y.; Patel, K.; Kong, I.; Whitehead, L.; Light, A.; Young, S.; Barrios, M.; Sargeant, C.; Rajasekhar, P.; Zhu, L.; Hempel, A.; Lin, A.; Rickard, J.; Hall, C.; Gangatirkar, P.; Yip, R.; Cawthorne, W.; Jacobsen, A.; Horne, C.; Ioannidis, L.; Hansen, D.; Day, J.; Wicks, I.; Law, C. W.; Ritchie, M. E.; Bowden, R.; Hildebrand, J.; O'Reilly, L.; Silke, J.; Giulino-Roth, L.; Tsui, E.; Rogers, K.; Hawkins, E.; Christensen, B.; Murphy, J. M.; Samson, A. L.

doi:10.1101/2023.10.31.565039

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bioRxiv · 10.1101/2023.10.31.565039

An immunohistochemical atlas of necroptotic pathway expression

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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. HighlightsO_LI13 automated immunohistochemistry protocols for detecting the necroptotic pathway C_LIO_LINecroptotic pathway expression is confined to fast-cycling immune barriers C_LIO_LINecroptotic pathway expression changes at sites of immunoinflammatory challenge C_LIO_LIImmunodetection of necrosomes in IBD patients is a putative new diagnostic tool C_LI

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Chiou, S., Al-Ani, A., Pan, Y., Patel, K., Kong, I., Whitehead, L., Light, A., Young, S., Barrios, M., Sargeant, C., Rajasekhar, P., Zhu, L., Hempel, A., Lin, A., Rickard, J., Hall, C., Gangatirkar, P., Yip, R., Cawthorne, W., Jacobsen, A., Horne, C., Ioannidis, L., Hansen, D., Day, J., Wicks, I., Law, C. W., Ritchie, M. E., Bowden, R., Hildebrand, J., O'Reilly, L., Silke, J., Giulino-Roth, L., Tsui, E., Rogers, K., Hawkins, E., Christensen, B., Murphy, J. M., Samson, A. L.. 2023-11-02. An immunohistochemical atlas of necroptotic pathway expression. https://doi.org/10.1101/2023.10.31.565039

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