{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T22:16:18Z","timestamp":1781648178124,"version":"3.54.5"},"reference-count":24,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T00:00:00Z","timestamp":1762128000000},"content-version":"vor","delay-in-days":2,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2025,11]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Skeletal muscle fiber type composition affects muscle function, metabolism, and disease vulnerability. In addition, muscle fiber type analysis informs disease diagnosis and underlying pathophysiology. Multiple methodologies can be used to assess muscle fiber type; however, immunofluorescence (IF) for myosin heavy chain (MyHC) isoforms is the most widely used modern approach due to its relative ease, time\u2010effectiveness, single\u2010cell resolution, and capacity to preserve spatial positioning within the native tissue architecture. Here, we present a protocol for IF for MyHC labeling on formalin\u2010fixed paraffin\u2010embedded (FFPE) mouse and human muscle sections. We then describe a modified procedure for fiber type analysis of the intact mouse lower hindlimb, enabling high\u2010throughput muscle composition and morphological analysis across distinct muscles on a single tissue section. Traditionally, IF labeling for MyHC isoforms required fresh tissue flash\u2010frozen in liquid nitrogen\u2013cooled isopentane, which, while effective, presents challenges for sample processing and preservation, long\u2010term storage, transport, and biosafety. Comparatively, embedding tissue in paraffin after formalin fixation streamlines clinical workflows, preserves morphology, improves long\u2010term sample stability, and simplifies sample storage and transport. Furthermore, FFPE effectively inactivates most infectious agents, which can be retained in frozen sections. Thus, FFPE samples are typically safe for standard laboratory handling and are not classified as biohazardous. This approach can be adapted for use with a range of downstream applications, including integration of fiber type analysis with emerging next\u2010generation techniques that favor FFPE samples. In sum, this method offers a robust alternative to traditional fresh\u2010frozen protocols and allows for simultaneous fiber type analysis across multiple muscle tissues. \u00a9 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol<\/jats:bold>\n                    : Multiplex Immunofluorescence for MyHC Labeling in FFPE Skeletal Muscle Tissues\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Alternate Protocol 1<\/jats:bold>\n                    : Immunofluorescence for MyHC 2x Labeling in FFPE Skeletal Muscle Tissues\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Alternate Protocol 2<\/jats:bold>\n                    : Multiplex Immunofluorescence for MyHC Labeling in FFPE Whole Hindlimb Sections\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70246","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T15:20:48Z","timestamp":1762183248000},"source":"Crossref","is-referenced-by-count":2,"title":["Adaptable Immunofluorescence Protocol for Muscle Fiber Typing in FFPE Human and Mouse Skeletal Muscle and Intact Mouse Hindlimbs"],"prefix":"10.1002","volume":"5","author":[{"given":"Connor","family":"Thomas","sequence":"first","affiliation":[{"name":"Department of Anatomy, Cell Biology, &amp; Physiology Indiana University School of Medicine, Indianapolis  Indianapolis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lainey M.","family":"Hibbard","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics Indiana University School of Medicine, Indianapolis  Indianapolis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth E.","family":"White","sequence":"additional","affiliation":[{"name":"Department of Medical and Molecular Genetics Indiana University School of Medicine, Indianapolis  Indianapolis"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2353-0325","authenticated-orcid":false,"given":"Steven S.","family":"Welc","sequence":"additional","affiliation":[{"name":"Department of Anatomy, Cell Biology, &amp; Physiology Indiana University School of Medicine, Indianapolis  Indianapolis"},{"name":"Indiana Center for Musculoskeletal Health Indiana University School of Medicine, Indianapolis  Indianapolis"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,11,3]]},"reference":[{"issue":"2","key":"e_1_2_8_2_1","first-page":"0","article-title":"Skeletal muscle fiber types in C57BL6J mice","volume":"21","author":"Augusto V.","year":"2017","journal-title":"Journal of Morphological Sciences"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/ijms26167986"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0035273"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmor.1052210207"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1093\/gerona\/58.11.B984"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1111\/sms.12185"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.5858\/2010\u20100702\u2010RAIR.1"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1093\/gerona\/50A.Special_Issue.11"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1111\/sms.14127"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1136\/jclinpath\u20102017\u2010204387"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1177\/32.6.6202737"},{"key":"e_1_2_8_13_1","volume-title":"Histological and histochemical methods","author":"Kiernan J.","year":"2015"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1152\/ajpendo.00512.2010"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1152\/japplphysiol.00624.2019"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.3390\/v7020666"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.14814\/phy2.15031"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1152\/ajpcell.00013.2006"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/1097\u20100029(20000915)50:6<500::AID\u2010JEMT7>3.0.CO;2\u20107"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1007\/s004180100268"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.3390\/sports9090127"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1152\/physrev.00031.2010"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1152\/ajpcell.1994.267.6.C1723"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1097\/MCO.0b013e328360272d"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1519\/JSC.0b013e318234eb6f"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70246","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T12:49:03Z","timestamp":1764938943000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70246"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11]]},"references-count":24,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2025,11]]}},"alternative-id":["10.1002\/cpz1.70246"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70246","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11]]},"article-number":"e70246"}}