For human brain sections, sections were deparaffinized, gradually rehydrated, and incubated for 10 min in 0.3% H2O2 diluted in methanol to quench endogenous peroxidase activity. For antigen retrieval, the sections were treated with 10 mM citric acid buffer (pH 6.0) and heated in a microwave oven. After three washes with PBS, the sections were incubated with an antibody directed against p-MLKL at a dilution of 1:50 and incubated overnight at 4°C. After three rinses with PBS, the sections were incubated with secondary antibody (EnVision+ System horseradish peroxidase, Dako) for 60 min at room temperature and subsequently exposed to the chromogen (3,3′ diaminobenzidine tetrachloride) system for 5 min. The sections were counterstained with hematoxylin, dehydrated, cleared, and cover-slipped. Slides were examined and captured the images by light microscopy (Leica, Germany). Signal analysis was done with Leica Q550CW image analysis system (Germany) and QWin software. Signal was not detected in the negative control experiments including secondary antibody alone. The experiment was carried out in a double-blinded manner.

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