Systematic Necropsy and Tissue Sampling in the Neotenic Axolotl (Ambystoma mexicanum)

Axolotls (Ambystoma mexicanum) are neotenic salamanders widely used as experimental models in regenerative biology, developmental research, toxicology, and pharmacology. Despite their extensive use in laboratory research and conservation programs, standardized necropsy procedures for this species remain limited and poorly documented. The absence of systematic protocols limits the consistency of postmortem examinations and complicates the interpretation of pathological findings in both experimental and diagnostic contexts. Here, we present a detailed and reproducible necropsy protocol for the neotenic axolotl based on 17 systematic necropsies performed under controlled laboratory conditions. The procedure describes step-by-step methods for external examination, access to the cranial cavity, exposure of the spinal cord, and systematic exploration of the coelomic cavity. Particular emphasis is placed on anatomical orientation, preservation of organ relationships, and strategies to minimize tissue damage during dissection. The protocol also includes guidelines for tissue sampling, fixation, and transport for downstream laboratory analyses such as histopathology and microbiology. Protocol refinement revealed practical considerations not previously described for this species, including variability in hepatic size and the absence of coelomic fat bodies in examined specimens, which simplified visceral exposure and influenced dissection strategy. These findings highlight the need to adapt necropsy techniques to species-specific anatomical characteristics. This protocol provides a practical reference for veterinarians, pathologists, and researchers working with A. mexicanum, facilitating standardized postmortem examinations and improving the reliability and reproducibility of diagnostic and research findings in this important amphibian model. © 2026 Wiley Periodicals LLC. Basic Protocol 1: External examination Basic Protocol 2: Organs of the head and spinal cord examination Basic Protocol 3: Organs of the coelomic cavity examination Support Protocol: Collection, preservation and transport of specimens for laboratory analysis.