An immunohistochemical atlas of necroptotic pathway expression Necroptosis is a regulated form of cell death involved in inflammatory diseases of the gut, skin, lung, and ischemic-reperfusion injuries of the kidney, heart, and brain. However, identifying cells and tissues undergoing necroptotic death in vivo has been challenging due to the lack of robust immunohistochemical detection protocols. This study provides automated immunohistochemistry protocols to detect core necroptosis regulators—Caspase-8, RIPK1, RIPK3, and MLKL—in formalin-fixed mouse and human tissues. The results show significant heterogeneity in protein expression, with short-lived immune barrier cells expressing necroptotic effectors, while long-lived cells lack RIPK3 or MLKL. Local changes in necroptotic effector expression occur in response to inflammation, dysbiosis, or immune challenge, consistent with necroptosis being dysregulated in disease contexts. The necroptotic pathway is well-defined and can be activated by various inflammatory cues. Caspase-8 is a critical negative regulator of necroptotic signaling, and its deletion promotes the oligomerization of RIPK1, TRIF, and/or ZBP1. The oligomeric structure, known as the necrosome, activates downstream effectors RIPK3 and MLKL. RIPK3 recruits MLKL to the necrosome, where it phosphorylates MLKL, leading to conformational changes, release from the necrosome, and trafficking to the plasma membrane. At the plasma membrane, accumulation of activated MLKL to a critical threshold level is required for membrane permeabilization, leading to cell death. The study developed automated immunohistochemistry protocols to detect Caspase-8, RIPK1, RIPK3, and MLKL in mouse formalin-fixed paraffin-embedded tissues. These protocols enabled the assembly of an atlas of necroptotic pathway expression in mouse tissues under basal conditions and during innate immune challenge. The necroptosis machinery is rarely expressed in cell types other than short-lived barrier cells, but sterile inflammation increased RIPK3 expression in the gut and liver, broadly predisposing multiple cell types to necroptotic death. In contrast, the elimination of the intestinal microflora diminished the expression of RIPK3 and MLKL to reduce necroptotic propensity in the gut. RIPK3 is also uniquely upregulated in splenic germinal centers, suggesting a non-necroptotic role in humoral immunity. The study also presented robust protocols for detecting human Caspase-8, RIPK1, RIPK3, and MLKL and illustrated their utility for detecting dysregulated necroptosis in biopsies from patients with inflammatory bowel disease (IBD). Collectively, these protocols will empower the definitive evaluation of where and when necroptosis occurs inAn immunohistochemical atlas of necroptotic pathway expression Necroptosis is a regulated form of cell death involved in inflammatory diseases of the gut, skin, lung, and ischemic-reperfusion injuries of the kidney, heart, and brain. However, identifying cells and tissues undergoing necroptotic death in vivo has been challenging due to the lack of robust immunohistochemical detection protocols. This study provides automated immunohistochemistry protocols to detect core necroptosis regulators—Caspase-8, RIPK1, RIPK3, and MLKL—in formalin-fixed mouse and human tissues. The results show significant heterogeneity in protein expression, with short-lived immune barrier cells expressing necroptotic effectors, while long-lived cells lack RIPK3 or MLKL. Local changes in necroptotic effector expression occur in response to inflammation, dysbiosis, or immune challenge, consistent with necroptosis being dysregulated in disease contexts. The necroptotic pathway is well-defined and can be activated by various inflammatory cues. Caspase-8 is a critical negative regulator of necroptotic signaling, and its deletion promotes the oligomerization of RIPK1, TRIF, and/or ZBP1. The oligomeric structure, known as the necrosome, activates downstream effectors RIPK3 and MLKL. RIPK3 recruits MLKL to the necrosome, where it phosphorylates MLKL, leading to conformational changes, release from the necrosome, and trafficking to the plasma membrane. At the plasma membrane, accumulation of activated MLKL to a critical threshold level is required for membrane permeabilization, leading to cell death. The study developed automated immunohistochemistry protocols to detect Caspase-8, RIPK1, RIPK3, and MLKL in mouse formalin-fixed paraffin-embedded tissues. These protocols enabled the assembly of an atlas of necroptotic pathway expression in mouse tissues under basal conditions and during innate immune challenge. The necroptosis machinery is rarely expressed in cell types other than short-lived barrier cells, but sterile inflammation increased RIPK3 expression in the gut and liver, broadly predisposing multiple cell types to necroptotic death. In contrast, the elimination of the intestinal microflora diminished the expression of RIPK3 and MLKL to reduce necroptotic propensity in the gut. RIPK3 is also uniquely upregulated in splenic germinal centers, suggesting a non-necroptotic role in humoral immunity. The study also presented robust protocols for detecting human Caspase-8, RIPK1, RIPK3, and MLKL and illustrated their utility for detecting dysregulated necroptosis in biopsies from patients with inflammatory bowel disease (IBD). Collectively, these protocols will empower the definitive evaluation of where and when necroptosis occurs in