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 13-galac- tosidase (SA-13Gal) and immunoperoxidase (IPO) activity in fresh -frozen murine tissues. We describe steps for tissue collection, solution preparation, SA-13Gal staining, IPO staining, hematoxylin counterstaining, microscopic observation, and signal quantification. This protocol can be used to detect in situ proteins alongside SA-13Gal activity. For complete details on the use and execution of this protocol, please refer to Pacheco -Rivera et al.1