Elastic Fiber Profiling in Lung Tissue Using Quantitative Imaging Analyses.

Remodeling of the pulmonary extracellular matrix (ECM) is a key feature of lung injury and disease. Elastic fibers are a central component of the lung ECM, providing resilience and recoil necessary for normal respiratory function, and their disruption contributes to impaired tissue mechanics. This protocol describes a robust, reproducible workflow for the preparation and analysis of murine lung tissue, with a focus on the visualization and quantitative profiling of elastic fibers. It outlines optimized tissue processing and histological staining methods to preserve and detect elastin structures. In addition, the protocol details an image analysis pipeline enabling quantitative assessment of elastic fiber abundance, organization, and spatial distribution within the lung. The workflow further incorporates stain vector estimation in QuPath, color deconvolution in Fiji/ImageJ, and TWOMBLI-based image analysis to enable quantitative assessment of elastic fiber abundance, branching, and network complexity. Strategies for image acquisition, region-of-interest selection, parameter optimization, and quality control are included to ensure reproducibility and minimize user-dependent bias. Using this workflow, we successfully identified alterations in elastic fiber organization and network architecture in the lungs of obese mice, demonstrating its applicability for detecting disease-associated ECM remodeling. Together, these approaches provide a comprehensive framework to investigate elastic fiber remodeling and its contribution to tissue architecture in physiological and pathological conditions. This method facilitates the study of fibroblast-driven structural changes and supports mechanistic insights into ECM alterations underlying lung disease.