Mikayla McWilliams, “Developing Protocol for Immunofluorescent Staining of Healing Murine Callus”
Mentor: Priyatha Premnath, Biomedical Engineering, Engineering & Applied Science (College of)
Poster #166
Immunofluorescent staining is a technique used to visualize and localize specific molecules within cells and tissues. This method employs antibody molecules to target particular proteins or antigens, providing detailed insights into protein distribution, cellular function, and potential drug interactions. In this experiment, two antibodies were used: DAPI, which binds to and stains double-stranded DNA (excited at 360 nm), and anti-alpha smooth muscle actin (α-SMA), which stains smooth muscle cells, including those in blood vessel walls and the gut (excited at 488 nm). A staining protocol was developed and perfected, including deparaffinization, rehydration, antigen retrieval, blocking, primary and secondary antibody incubation, and final mounting, storage, and imaging. This protocol enables the visualization and localization of both nuclei and blood vessels using the Zeiss LSM 710 Confocal Microscope. The results can be compared to processed CT scans of the same bone to assess the alignment of blood vessel localization, as well as nuclei distribution. These findings support our lab’s developing theory that mechanical stress influences vascularization during murine callus healing, providing a base for future studies on bone regeneration and repair.