This study presents an automated immunohistochemical atlas of necroptotic pathway expression in mouse and human tissues. Necroptosis, a form of regulated cell death, is implicated in various inflammatory diseases but its precise identification in vivo has been challenging due to the lack of robust detection methods. The authors developed and validated automated immunohistochemistry protocols to detect key necroptotic regulators—Caspase-8, RIPK1, RIPK3, and MLKL—in formalin-fixed paraffin-embedded mouse tissues. They found that the necroptotic pathway is predominantly expressed in short-lived immune barrier cells, with surprising heterogeneity in protein expression. Local changes in necroptotic effector expression occur in response to insults such as inflammation, dysbiosis, or immune challenge, suggesting dysregulation in disease contexts. The methods will facilitate the precise localization and evaluation of necroptotic signaling in vivo. The study also explored the utility of these protocols in human tissues, particularly in inflammatory bowel disease (IBD), and identified potential non-necrotic roles for RIPK3 in adaptive immunity. Overall, the optimized immunohistochemistry protocols provide a valuable tool for studying necroptosis in health and disease.This study presents an automated immunohistochemical atlas of necroptotic pathway expression in mouse and human tissues. Necroptosis, a form of regulated cell death, is implicated in various inflammatory diseases but its precise identification in vivo has been challenging due to the lack of robust detection methods. The authors developed and validated automated immunohistochemistry protocols to detect key necroptotic regulators—Caspase-8, RIPK1, RIPK3, and MLKL—in formalin-fixed paraffin-embedded mouse tissues. They found that the necroptotic pathway is predominantly expressed in short-lived immune barrier cells, with surprising heterogeneity in protein expression. Local changes in necroptotic effector expression occur in response to insults such as inflammation, dysbiosis, or immune challenge, suggesting dysregulation in disease contexts. The methods will facilitate the precise localization and evaluation of necroptotic signaling in vivo. The study also explored the utility of these protocols in human tissues, particularly in inflammatory bowel disease (IBD), and identified potential non-necrotic roles for RIPK3 in adaptive immunity. Overall, the optimized immunohistochemistry protocols provide a valuable tool for studying necroptosis in health and disease.