Expansion microscopy has enabled super resolution imaging of biological samples. The accurate measurement of expansion factor and distortion typically requires locating and imaging the same region of interest in the sample before and after expansion, which is often time-consuming to achieve. Here we introduce a convenient method for relocation by utilizing isolated porcine glomeruli as landmarks during expansion. Following heat denaturation and proteinase K digestion protocols, the glomeruli exhibit expansion factor of 3.5 to 4 (only 7%-16% less expanded than the hydrogel), and 1% to 2% of relative distortion.Due to its appropriate size of 100 to 300 μm, the location of the glomerulus in the sample are visible to eyes, while its detailed shape only requires bright field microscopy. For expansion factors ranging from 3 to 10, the region in the vicinity of the glomerulus can be easily re-identified, and sometimes allows quantification of expansion factor and distortion under bright field without fluorescent labels.
Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, and progressive neurodegenerative disorder with no cure. Cu/Zn-superoxide dismutase (SOD1) was the first identified protein associated with familial ALS; and aggresome formation of misfolded SOD1 is closely associated with ALS pathogenesis. HDAC6, one of the histone deacetylase family members, has already been demonstrated to play an important role in regulating aggresome formation of misfolded proteins and protecting cells against the toxicity induced by misfolded proteins. In this study, we found that in a cellular model with impaired proteasome activity, the TAR DNA-binding protein 43, which is closely linked with ALS and associated with various neurodegenerative disorders such as frontotemporal lobar degeneration, Alzheimer's disease, and Parkinson's disease, can regulate mutant SOD1 aggresome formation through an HDAC6-dependent manner. TDP-43 deficiency did not affect poly-ubiquitination of mutant SOD1, whereas it greatly decreased the expression level of HDAC6, which is required for aggresome formation of ALS-linked mutant SOD1. Moreover, overexpression of siRNA-resistant HDAC6 restored mutant SOD1 aggresome formation in TDP-43knockdown cells. Thus, our data provide evidence that TDP-43 plays an important role in mutant SOD1 aggresome formation through its regulation of HDAC6.
Expansion microscopy allows super resolution imaging of cellular structures by fluorescence microscopy. However, current protocols achieving large expansion factors (near 8 and beyond), are only applicable to cultured cells and thin tissue slices, but not to bulk tissue in general. Here, we present a method that allows unlimited cycles of expansion of bulk tissue with high isotropy, which we term as Cyc-ExM. The protocol uses identical gel recipe and denaturation reagents in each expansion cycle, which provides ease to the procedure. Cyc-ExM expands biological tissues ~9 fold in each dimension from 7 cycles of expansion and 17-27 fold from 10 cycles. The covalent staining procedure was also optimized to reveal ultrastructural features. Besides, Cyc-ExM is compatible with antibody labeling and multi-scale 3D imaging. Despite volume dilution of signal during expansion, we developed a signal amplification method achieving 8-fold increase in signal, which greatly facilitates the resolution of sub-organelle structures within various tissues.
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