{"doi":"10.1016/j.xpro.2024.103549","title":"Protocol for the three-dimensional analysis of rodent skeletal muscle","abstract":"Confocal imaging is a powerful tool capable of analyzing cellular spatial data within a given tissue. Here, we present a protocol for preparing optically cleared extensor digitorum longus (EDL) skeletal muscle samples suitable for confocal imaging/computational analysis. We describe steps for sample preparation (including perfusion fixation and tissue clearing of muscle samples), image acquisition, and computational analysis, with sample segmentation/3D rendering outlined. This protocol can be applied to characterize various cell types, including muscle satellite cells (muscle stem cells) and capillary endothelial cells within rodent skeletal muscle. For complete details on the use and execution of this protocol, please refer to Verma et al., 1 Verma et al., 2 Karthikeyan et al., 3 and Karthikeyan et al. 4 • 3D imaging analysis for muscle stem cells and capillaries in mouse skeletal muscle • Tissue clearing techniques for mouse skeletal muscle samples • 3D skeletal muscle image acquisition by confocal microscope • Data analysis by using Fiji/Ilastik and Imaris software for 3D image analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Confocal imaging is a powerful tool capable of analyzing cellular spatial data within a given tissue. Here, we present a protocol for preparing optically cleared extensor digitorum longus (EDL) skeletal muscle samples suitable for confocal imaging/computational analysis. We describe steps for sample preparation (including perfusion fixation and tissue clearing of muscle samples), image acquisition, and computational analysis, with sample segmentation/3D rendering outlined. This protocol can be applied to characterize various cell types, including muscle satellite cells (muscle stem cells) and capillary endothelial cells within rodent skeletal muscle.","journal":"STAR Protocols","year":2025,"id":538442,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.8885,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":587983,"name":"Yoko Asakura","orcid":"0000-0003-4107-4236","position":1,"is_corresponding":false},{"id":1164524,"name":"Mayank Verma","orcid":"0000-0003-0167-0842","position":2,"is_corresponding":false},{"id":587984,"name":"Atsushi Asakura","orcid":"0000-0001-8078-1027","position":3,"is_corresponding":false},{"id":1425414,"name":"Smrithi Karthikeyan","orcid":null,"position":0,"is_corresponding":true}],"reference_count":14,"raw_metadata":null,"created_at":"2026-07-19T02:52:21.389196Z","pmid":"39798097","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}