Protocol to detect senescence-associated β-galactosidase and immunoperoxidase activity in fresh-frozen murine tissues

Double labeling to identify different markers in the same tissue section represents a useful tool either for in situ diagnosis or characterization of molecular associations. Here, we present a protocol to detect senescence-associated β-galactosidase (SA-βGal) and immunoperoxidase (IPO) activity in fresh-frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-βGal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-βGal activity. For complete details on the use and execution of this protocol, please refer to Pacheco-Rivera et al.¹.