Standardization of Immunohistochemistry Using an Anti-WASLB Antibody in the Skeletal Muscle of Teleost Fish

Immunohistochemistry (IHC) is a widely used technique for the identification, localization, and analysis of protein expression in tissues, playing a critical role in pathological diagnosis and validation of data derived from molecular biology. Despite its well-established use in mammalian tissues, applying IHC to fish still presents challenges, particularly due to the use of heterologous antibodies and the presence of paralogous genes. In this context, the present study aimed to standardize an immunohistochemistry protocol adapted for fish skeletal muscle using a biotinylated monoclonal antibody designed against a conserved region of the waslb gene for pacu (Piaractus mesopotamicus) and zebrafish (Danio rerio). Muscle samples were fixed in 4% paraformaldehyde, embedded in paraffin, and subjected to heat-induced antigen retrieval. After blocking nonspecific binding sites, histological sections were incubated with the primary antibody at an optimized 1:50 dilution, followed by detection with fluorophore-conjugated streptavidin (1:200) and nuclear counterstaining with DAPI. Reaction specificity was assessed using negative controls with a 20,000-fold molar excess of the immunizing peptide. The standardized protocol resulted in specific and reproducible labeling of the waslb protein in the skeletal muscle of both species, with no signal observed in negative controls; however, it is important to emphasize that this protocol can also be applied to antibodies against other fish genes. Thus, this method represents a reliable tool for protein analysis in fish muscle tissues, contributing to physiological, experimental, and comparative studies in non-mammalian models. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Immunohistochemical reaction against the WASLB antibody in paraffin-embedded skeletal muscle sections of pacus and zebrafish.