{"title":"Expansion Microscopy of the Enteric Nervous System: A Feasibility Study.","authors":"Xin Xu, Wenchuan Zhang, Menachem Hanani","doi":"10.3390/cells14181463","DOIUrl":null,"url":null,"abstract":"<p><p>Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3-5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X-Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":"14 18","pages":""},"PeriodicalIF":5.2000,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12468614/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cells","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.3390/cells14181463","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Expansion microscopy (ExM) enables conventional light microscopes to achieve nanoscale resolution by physically enlarging biological specimens. While ExM has been widely applied in neurobiology, it has not been adapted for the enteric nervous system (ENS). Here, we provide a detailed and reproducible protocol for applying ExM to mouse colonic ENS tissue. The procedure includes preparation of the external muscle layers with the myenteric plexus, histochemical staining for NADPH-diaphorase, immunostaining for glial fibrillary acidic protein (GFAP), anchoring of biomolecules, gelation, proteinase K digestion, and isotropic expansion in a swellable polymer matrix. Step-by-step instructions, required reagents, and critical parameters are described to ensure robustness and reproducibility. Using this protocol, tissues expand 3-5-fold, allowing neuronal somata, fibers, and glial cell processes to be clearly visualized by standard brightfield or fluorescence microscopy. The tissue architecture is preserved, with distortion in the X-Y plane of about 7%. This protocol provides a reliable framework for high-resolution structural analysis of the ENS and can be readily adapted to other peripheral tissues.
CellsBiochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
9.90
自引率
5.00%
发文量
3472
审稿时长
16 days
期刊介绍:
Cells (ISSN 2073-4409) is an international, peer-reviewed open access journal which provides an advanced forum for studies related to cell biology, molecular biology and biophysics. It publishes reviews, research articles, communications and technical notes. Our aim is to encourage scientists to publish their experimental and theoretical results in as much detail as possible. There is no restriction on the length of the papers. Full experimental and/or methodical details must be provided.