Immunofluorescence Staining on Cryosections with High Autofluorescence in African Killifish

The African killifish Nothobranchius furzeri has been considered as an attractive model for exploring biological questions related to regeneration, aging, and human diseases. Characterization of gene expression and localization in cells or tissues is a routine work in many fields of biology, which facilitates the understanding of gene functions in a specific biological context. Diverse histology-related techniques have been developed to fulfill this goal including one of the most popular methodologies, the immunofluorescence staining on cryosections. Such technique has been widely applicated in many studies in African killifish to examine spatiotemporal pattern of proteins. However, many tissues (e.g., brain, spinal cord, tail, and heart) of African killifish have relatively high autofluorescence (AF), which makes the application of immunofluorescence staining more challenging. Here, we take advantage of bleaching and commercially available autofluorescence quenching reagents to overcome problems caused by autofluorescence. This chapter describes a detailed and reproducible protocol for immunofluorescence staining on cryosections in African killifish. The reliability of this protocol has been demonstrated by the successful immunostaining of glial fibrillary acidic protein (GFAP), Sox2, α-tubulin, and Dach1 antibodies.