Conventional immunostaining protocols often lack versatility, catering predominantly to model organisms and failing to accommodate diverse specimens, particularly those that are non-perfused and are over-fixed. This chapter introduces an innovative immunofluorescence protocol designed to localize protein targets in brain tissue from a wide spectrum of mammals, spanning both model and non-model species. This method widely applies to label astrocytes from diverse mammalian species; however, it is usable to immunostain other protein targets. We provide a comprehensive guide encompassing the preparation of fresh and fixed tissues, incorporating essential steps such as deparaffinization, fixation, and cryoprotection. The immunofluorescence procedures outlined cover crucial aspects including antigen retrieval, mitigation of autofluorescence, nuclear staining, and meticulous mounting techniques, culminating in detailed instructions for image collection and resolutions for possible problems that could be encountered. By offering adaptable methodologies tailored to the complexities of diverse mammalian brain tissues, this chapter aims to advance the application of immunofluorescence techniques in astrocyte research and neurobiological research at large.