Protocol for patterning competency assay in regenerative axolotl limb cells using the competency accessory limb model

A key question in limb regeneration is how axolotl limb cells become competent to specific types of regeneration signals. Here, we present the competency accessory limb model (CALM), a regenerative assay that assesses whether limb cells have achieved the ability to respond to patterning signals. We describe the steps for positioning a wound, deviating the nerve into a limb wound site, and injecting retinoic acid. We also detail the harvest and analyses in both anterior and posterior CALMs. For complete details on the use and execution of this protocol, please refer to Raymond et al.¹.