Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures.
Ewa LangnerPongpratch PuapatanakulRachel PudlowskiDema Yaseen AlsabbaghJeffrey H MinerAmjad HoraniSusan K DutcherSteven L BrodyJennifer T WangHani Y SuleimanMoe R MahjoubPublished in: bioRxiv : the preprint server for biology (2024)
Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.
Keyphrases
- high resolution
- extracellular matrix
- single molecule
- single cell
- endothelial cells
- cell therapy
- randomized controlled trial
- high speed
- mass spectrometry
- physical activity
- induced pluripotent stem cells
- label free
- mental health
- minimally invasive
- atomic force microscopy
- mesenchymal stem cells
- pluripotent stem cells
- fluorescence imaging