Antigen retrieval-immunofluorescence on free floating sections to visualize the liver lobule and its cellular makeup

Tissue histology is widely performed on one cell-thick sections. While classical histology has helped establish the fundamentals of tissue organization and function, the lack of three dimensionality has hindered holistic analyses. A comprehensive view has been particularly challenging to attain in organs with high cellularity. Recently, the use of vibratome-generated thick sections has been on the rise, offering an opportunity to study spatial architecture. However, the range of viable antibodies has been limiting. Here, we have developed a mechanical stabilization method to enable antigen retrieval on thick vibratome sections for difficult antigens while maintaining tissue integrity. This cost-efficient method widens the utility of thick vibratome sections and facilitates holistic analysis of complex tissues.Method Summary Paraformaldehyde-fixed mouse liver samples are sectioned on a vibratome at 100 µm. The free-floating sections are permeabilized, followed by exposure to one of two heat-induced antigen retrieval methods selected based on the antigen of interest. Antigen retrieval is carried out in a mechanical stabilization apparatus developed here to prevent free-floating sections from deforming. The immunostaining is then carried out with noted modifications to previously established protocols.Multidisciplinary Abstract Spatial organ biology widely relies on slicing tissues into one cell-thick sections. These studies have advanced our understanding of organ structure and function; yet, thin sections have been insufficient to investigate three-dimensional organization in complex organs. A more recent approach utilizes thicker sections, enabling visualization of volumetric tissue architecture. Currently, few protocols exist to strengthen immunofluorescence signal on thick tissue sections while maintaining tissue integrity, particularly for difficult antigens. Here, we developed a simple, affordable method that mechanically stabilizes thick tissue sections and preserves tissue integrity during heat-mediated antigen retrieval used to enhance antigen accessibility. This method widens the utility of thick tissue sections and facilitates holistic analysis of complex tissues, opening new possibilities for spatial organ research.