Biomechanical Studies with Xenopus Animal Caps: A 3D-Printed Mechanical Bracket for Maintaining Tissue Stretch During Immunofluorescence Analysis.

Mechanical forces are central regulators of embryonic morphogenesis, influencing cell division, migration, and fate decisions. The amphibian model Xenopus laevis provides a tractable system for dissecting mechanotransduction pathways, yet existing approaches to apply reproducible force to animal cap tissues are limited by reliance on expensive custom-built devices and the inability to preserve tissue deformation during multiday immunofluorescence protocols. Here, we present a method for applying and maintaining stretch in X. laevis animal caps using a low-cost, 3D-printed mechanical bracket. This device locks explants in a stretched state throughout fixation, staining, clearing, and imaging, thereby maintaining key features, such as cell geometry, which are lost if the stretched state is released. We describe the design and assembly of the bracket, preparation of elastomeric Polydimethylsiloxane (PDMS) membranes, embryo collection and dissection, and protocols for stretching, fixation, and volumetric immunofluorescence imaging. This approach enables high-resolution analysis of force-dependent molecular and cellular processes, while lowering technical barriers for mechanobiology research. By preserving tissue geometry during complex workflows, the method expands the utility of X. laevis animal caps as a versatile platform for studying how mechanical cues shape developing tissues.