Immunolocalization in Xenopus.

Immunolocalization is an essential technique for studying protein distribution in Xenopus species, enabling researchers to map endogenous protein expression during development and analyze responses to various treatments. This technique is also useful to detect epitope-tagged proteins, tagged morpholinos, and tracers. Although antibodies raised specifically against Xenopus are preferred for the detection of endogenous proteins, the limited availability of these reagents implies that cross-species antibodies are frequently employed and remain a crucial strategy. Consequently, rigorous antibody selection and validation are essential for ensuring the success and reliability of the technique. This chapter details immunohistochemistry (IHC) and immunofluorescence (IF) protocols that can be seamlessly combined with whole-mount in situ hybridization (WISH) techniques to simultaneously detect both protein and mRNA localization in Xenopus. We also provide comprehensive guidance on antibody selection criteria, specificity testing, and subsequent IF quantitative image analysis, which allows for robust statistical comparison of fluorescence differences across samples, regions of interest, or subcellular domains.