Ultrastructure expansion microscopy (U‐ExM) of mouse and human kidneys for analysis of subcellular structures

Abstract

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 embedding conditions. Overall, this protocol provides a simple and cost‐effective approach to analyze both preclinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Mapping protein distribution in the canine photoreceptor sensory cilium and calyceal processes by ultrastructure expansion microscopy

“…Here, we demonstrated that the duration of PFA fixation and long- term storage at -80°C do not significantly impact the degree of retinal tissue expansion, achieving approximately 4x isotropic expansion ( Figure 2 ). This observation aligns with the recent reports that applied U-ExM to PFA-fixed frozen and formalin-fixed paraffin-embedded mouse tissues [20, 28]. Notably, our results showed that U-ExM enabled super-resolution imaging of archival samples that had been cryopreserved for over a decade, providing comparable imaging quality to that obtained from fresh samples.…”

“…Based on these findings, we assumed that the region with glutamylation outside the tubulin axoneme is equivalent to the CC and measured the length and width of each compartment of the PSC axoneme ( Figure 3E ). As previously reported for other cell types [27, 28], the length of the MC was significantly longer than that of the daughter centriole (DC) in each photoreceptor subtype. More interestingly, cone photoreceptors had notably longer BBs than rods (Rod DC, 269.71 ± 24.00 μm; Cone DC, 357.27 ± 37.26 μm; Rod MC, 372.06 ± 55.22 μm; Cone MC, 465.84 ± 40.58 μm; Figure 3F ).…”