Background

Whole-mount immunohistochemistry, a technique that combines the visualization of tissue structures with the specific detection of proteins using antibodies, can be traced back to the studies by Albert Coons and colleagues in 1941 [1]. Over the years, this technique has been widely used in many research laboratories (especially in developmental biology and neuroscience studies) as well as in clinical diagnostics [2,3]. In general, it can be applied to whole zygotic embryos as well as to dissected tissues, with fixation and preservation of tissue integrity [4], permeabilization to allow antibody access [5], and staining with specific probes [6,7] playing key roles in the successful application of this technique. Yet, despite significant developments and refinements, particularly in sample preparation and staining procedures, the selection of the most suitable method must be based on parameters such as the type of specimen under investigation and the degree of sensitivity required.

In different research areas, zebrafish are increasingly employed as model systems to investigate molecular, genetic, and cellular aspects of developmental and neurobiological processes due to several advantages over other animal models [8,9]. However, although zebrafish represent an excellent experimental system, protein detection methods must be carefully adjusted to account for factors such as tissue permeability, pigmentation, and developmental stage [10,11]. Indeed, there is no single protocol suitable for all experimental conditions, as the effective application of a specific working procedure depends strongly on the antigen–antibody combination [12,13].

Several alternative methodologies have been used to assess protein expression, including section-based immunohistochemistry, in situ hybridization, and tissue-clearing approaches coupled with advanced imaging techniques [14–16]. In addition, tissue- and cell-specific visualization in zebrafish is commonly achieved using transgenic reporter lines or chemical labeling techniques [17,18]. However, their use is limited by the availability of suitable promoters, variability in expression levels, and the need for specialized genetic tools and breeding strategies. Similarly, chemical approaches are often restricted to specific tissues or structures and may lack cellular resolution or specificity for particular protein targets. In contrast, whole-mount immunohistochemistry allows direct detection of endogenous proteins with cellular resolution, without the need for genetic modification. However, these techniques generally require specialized reagents, longer processing times, and access to advanced microscopy platforms. Therefore, in this chapter, a step-by-step protocol for the localization of tyrosine hydroxylase (TH), a standard marker of dopaminergic neurons in the central nervous system [19], in zebrafish larvae is presented. This protocol is adapted from previously published work [20] and includes critical steps and practical considerations to ensure reliable and reproducible staining. Compared with other published whole-mount approaches, the protocol described here emphasizes optimized permeabilization and bleaching steps to improve antibody penetration and minimize pigmentation-related background, making it particularly suitable for early larval stages. However, limitations include dependence on antibody quality and reduced applicability to later developmental stages or adult tissues without further protocol modification. Furthermore, despite recent advancements, the availability of validated antibodies in zebrafish remains limited, particularly for specific cell types or developmental stages, which can constrain immunohistochemical applications.

Beyond the assessment of dopaminergic neurons, this protocol can be readily adapted for the detection of additional neuronal or non-neuronal markers and applied in neurodevelopmental, neurotoxicity, and pharmacological studies using zebrafish larvae.