--- _id: '6052' abstract: - lang: eng text: 'Expansion microscopy is a relatively new approach to super-resolution imaging that uses expandable hydrogels to isotropically increase the physical distance between fluorophores in biological samples such as cell cultures or tissue slices. The classic gel recipe results in an expansion factor of ~4×, with a resolution of 60–80 nm. We have recently developed X10 microscopy, which uses a gel that achieves an expansion factor of ~10×, with a resolution of ~25 nm. Here, we provide a step-by-step protocol for X10 expansion microscopy. A typical experiment consists of seven sequential stages: (i) immunostaining, (ii) anchoring, (iii) polymerization, (iv) homogenization, (v) expansion, (vi) imaging, and (vii) validation. The protocol presented here includes recommendations for optimization, pitfalls and their solutions, and detailed guidelines that should increase reproducibility. Although our protocol focuses on X10 expansion microscopy, we detail which of these suggestions are also applicable to classic fourfold expansion microscopy. We exemplify our protocol using primary hippocampal neurons from rats, but our approach can be used with other primary cells or cultured cell lines of interest. This protocol will enable any researcher with basic experience in immunostainings and access to an epifluorescence microscope to perform super-resolution microscopy with X10. The procedure takes 3 d and requires ~5 h of actively handling the sample for labeling and expansion, and another ~3 h for imaging and analysis.' article_processing_charge: No article_type: original author: - first_name: Sven M full_name: Truckenbrodt, Sven M id: 45812BD4-F248-11E8-B48F-1D18A9856A87 last_name: Truckenbrodt - first_name: Christoph M full_name: Sommer, Christoph M id: 4DF26D8C-F248-11E8-B48F-1D18A9856A87 last_name: Sommer orcid: 0000-0003-1216-9105 - first_name: Silvio O full_name: Rizzoli, Silvio O last_name: Rizzoli - first_name: Johann G full_name: Danzl, Johann G id: 42EFD3B6-F248-11E8-B48F-1D18A9856A87 last_name: Danzl orcid: 0000-0001-8559-3973 citation: ama: Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. A practical guide to optimization in X10 expansion microscopy. Nature Protocols. 2019;14(3):832–863. doi:10.1038/s41596-018-0117-3 apa: Truckenbrodt, S. M., Sommer, C. M., Rizzoli, S. O., & Danzl, J. G. (2019). A practical guide to optimization in X10 expansion microscopy. Nature Protocols. Nature Publishing Group. https://doi.org/10.1038/s41596-018-0117-3 chicago: Truckenbrodt, Sven M, Christoph M Sommer, Silvio O Rizzoli, and Johann G Danzl. “A Practical Guide to Optimization in X10 Expansion Microscopy.” Nature Protocols. Nature Publishing Group, 2019. https://doi.org/10.1038/s41596-018-0117-3. ieee: S. M. Truckenbrodt, C. M. Sommer, S. O. Rizzoli, and J. G. Danzl, “A practical guide to optimization in X10 expansion microscopy,” Nature Protocols, vol. 14, no. 3. Nature Publishing Group, pp. 832–863, 2019. ista: Truckenbrodt SM, Sommer CM, Rizzoli SO, Danzl JG. 2019. A practical guide to optimization in X10 expansion microscopy. Nature Protocols. 14(3), 832–863. mla: Truckenbrodt, Sven M., et al. “A Practical Guide to Optimization in X10 Expansion Microscopy.” Nature Protocols, vol. 14, no. 3, Nature Publishing Group, 2019, pp. 832–863, doi:10.1038/s41596-018-0117-3. short: S.M. Truckenbrodt, C.M. Sommer, S.O. Rizzoli, J.G. Danzl, Nature Protocols 14 (2019) 832–863. date_created: 2019-02-24T22:59:20Z date_published: 2019-03-01T00:00:00Z date_updated: 2023-08-24T14:48:33Z day: '01' ddc: - '570' department: - _id: JoDa - _id: Bio doi: 10.1038/s41596-018-0117-3 ec_funded: 1 external_id: isi: - '000459890700008' pmid: - '30778205' file: - access_level: open_access checksum: 7efb9951e7ddf3e3dcc2fb92b859c623 content_type: application/vnd.openxmlformats-officedocument.wordprocessingml.document creator: kschuh date_created: 2021-06-29T14:41:46Z date_updated: 2021-06-29T14:41:46Z file_id: '9619' file_name: 181031_Truckenbrodt_ExM_NatProtoc.docx file_size: 84478958 relation: main_file success: 1 file_date_updated: 2021-06-29T14:41:46Z has_accepted_license: '1' intvolume: ' 14' isi: 1 issue: '3' language: - iso: eng month: '03' oa: 1 oa_version: Submitted Version page: 832–863 pmid: 1 project: - _id: 260C2330-B435-11E9-9278-68D0E5697425 call_identifier: H2020 grant_number: '754411' name: ISTplus - Postdoctoral Fellowships - _id: 265CB4D0-B435-11E9-9278-68D0E5697425 call_identifier: FWF grant_number: I03600 name: Optical control of synaptic function via adhesion molecules publication: Nature Protocols publication_status: published publisher: Nature Publishing Group quality_controlled: '1' scopus_import: '1' status: public title: A practical guide to optimization in X10 expansion microscopy type: journal_article user_id: 4359f0d1-fa6c-11eb-b949-802e58b17ae8 volume: 14 year: '2019' ...