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Recently, it has become evident that macrophage diversity increases in the liver during the pathogenesis of non-alcoholic steatohepatitis (NASH). Here, we provide a detailed protocol for the analysis of liver macrophage subsets in mice with non-alcoholic fatty liver disease (NAFLD) and early NASH using flow cytometry and immunofluorescence (IF). These methods can be used to assess the composition and localization of macrophage subsets during NASH. For complete details on the use and execution of this protocol, please refer to Daemen et a