A Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle to Visualize Endplates in Their Tissue Context

Williams, Marion Patrick Ivey; Rigon, Matteo; Straka, Tatjana; Hörner, Sarah Janice; Thiel, Manfred; Gretz, Norbert; Hafner, Mathias; Reischl, Markus; Rudolf, Rüdiger

doi:10.3389/fncel.2019.00049

Cited by 32 publications

(37 citation statements)

References 35 publications

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“…We are aware of only one previous report that observes myofiber loss in mdx, but it is not quantified 54 . www.nature.com/scientificreports/ In order to assess myofiber loss in mdx from a new perspective we utilized a variation of the MYOCLEAR technique to clear skeletal muscles and stain AChRs with fluorescent α-bungarotoxin 21 . This allowed us to assess the number of synapses in muscle tissue, extending the technique used by Faber et al into whole muscles 18 .…”

Section: Discussionmentioning

confidence: 99%

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss

Massopust

Lee

Pritchard

et al. 2020

Sci Rep

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The muscular dystrophy X-linked mouse (mdx) is the most commonly used preclinical model for Duchenne muscular dystrophy. Although disease progression in the mouse does not perfectly model the human disease, it shares many pathological features. Early characterizations of the model reported severe pathology through early adulthood followed by disease stabilization. As a result, research in the mdx mouse has largely focused on early adulthood. The overarching goal of this study is to improve the understanding of the mdx mouse model by tracking pathological features of the disease throughout life. We performed a thorough characterization of myofiber pathology in mdx mice from 2 weeks to 2 years of age. We report that individual mdx muscle fibers undergo progressive hypertrophy that continues through the lifespan. Despite massive hypertrophy on the myofiber level, we report no hypertrophy on the muscle level. These seemingly contradictory findings are explained by previously underappreciated myofiber loss in mdx mice. We conclude that due to myofiber loss, in combination with the progressive nature of other pathological features, aged mdx muscle tissue provides reliable benchmarks for disease progression that may be valuable in testing the efficacy of therapeutics for Duchenne muscular dystrophy.

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Section: Discussionmentioning

confidence: 99%

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss

Massopust

Lee

Pritchard

et al. 2020

Sci Rep

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“…27 But the thickness of fascia surrounding the muscle increased and the tissues become denser with age, the antibody penetration into muscles of adult mice is more difficult than that of young mice. The modified protocol described in this work achieved whole-mount labeling of adult tibialis anterior (about 3-mm thick), which is thicker than muscles such as extensor digitorum longus 28 and lumbricales. 29 It is supposed that the pretreatment of ScaleCUBIC-1 reagent in the m-iDISCO method might eliminate hindrance of fascia and could facilitate antibody penetration.…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method

Zhu

et al. 2020

Neurophoton.

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Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…Embedding in PBS or Mowiol (Sigma-Aldrich) and optical clearing with Glycerol: after immunofluorescence and 24 h prior to imaging, samples were mounted in 18 well µ-slides (ibidi) in either PBS or Mowiol. Glycerol-based RI matching was performed according to Williams et al (2019) by immersion of stained spheroids in an aqueous solution of 88% Glycerol (RI 1.459) ON at RT with gentle shaking, followed by mounting on 18 well µ-slides in the same solution. All procedures were executed with minimal exposure to light.…”

Section: Optical Clearingmentioning

confidence: 99%

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward

Nürnberg

Vitacolonna

Klicks

et al. 2020

Front. Mol. Biosci.

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Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid-and chip-based threedimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-tonoise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols-in particular, for screening purposes-clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

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A Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle to Visualize Endplates in Their Tissue Context

Cited by 32 publications

References 35 publications

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss

Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward

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