{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T02:13:52Z","timestamp":1790820832485,"version":"4.1.0"},"reference-count":25,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2022,9,21]],"date-time":"2022-09-21T00:00:00Z","timestamp":1663718400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Helmholtz Association"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering"],"abstract":"<jats:p>Histochemical staining of paraffin-embedded decalcified bone samples is commonly used in preclinical research of musculoskeletal diseases, enabling the visualization of multiple tissue components by the application of chromogens. The purpose of this study was to introduce a novel multicolor staining protocol involving optimized chemical reagents and procedure, allowing the identification of high-mineralized bone, low-mineralized fracture callus, cartilage and skeletal muscle fibers simultaneously. Fractured femur and healthy tail vertebra samples from adult male Sprague\u2013Dawley rats were decalcified with EDTA and formic acid, respectively, followed by paraffin embedding, tissue sectioning and multicolor staining. Conventional Movat\u2019s pentachrome and safranin O \/ fast green staining were conducted in parallel for comparison. Immunohistochemical staining of collagen type-X and micro-CT analysis were included to further validate the efficacy of the staining method. The multicolor staining allowed visualization of major musculoskeletal tissue components in both types of decalcified samples, providing quality outcomes with fewer chemical reagents and simplified procedures. Immunohistochemical staining demonstrated its capacity for identification of the endochondral ossification process during fracture healing. Micro-CT imaging validated the staining outcome for high-mineralized skeletal tissue. The application of the multicolor staining may facilitate future preclinical research involving decalcified paraffin-embedded samples.<\/jats:p>","DOI":"10.3390\/bioengineering9100488","type":"journal-article","created":{"date-parts":[[2022,9,21]],"date-time":"2022-09-21T21:22:23Z","timestamp":1663795343000},"page":"488","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Multicolor Histochemical Staining for Identification of Mineralized and Non-Mineralized Musculoskeletal Tissue: Immunohistochemical and Radiological Validation in Decalcified Bone Samples"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8330-942X","authenticated-orcid":false,"given":"Yu","family":"Sun","sequence":"first","affiliation":[{"name":"Institute of Metallic Biomaterials, Helmholtz-Zentrum Hereon, 21502 Geesthacht, Germany"},{"name":"Department of Orthopaedics, First Hospital of China Medical University, Shenyang 110001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6457-3607","authenticated-orcid":false,"given":"Heike","family":"Helmholz","sequence":"additional","affiliation":[{"name":"Institute of Metallic Biomaterials, Helmholtz-Zentrum Hereon, 21502 Geesthacht, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Regine","family":"Willumeit-R\u00f6mer","sequence":"additional","affiliation":[{"name":"Institute of Metallic Biomaterials, Helmholtz-Zentrum Hereon, 21502 Geesthacht, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1532","DOI":"10.1039\/D2BM00051B","article-title":"Dynamic in vivo monitoring of fracture healing process in response to magnesium implant with multimodal imaging: Pilot longitudinal study in a rat external fixation model","volume":"10","author":"Sun","year":"2022","journal-title":"Biomater. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"179","DOI":"10.22203\/eCM.v042a14","article-title":"Small-sized magnesium cylinders influence subchondral bone quality in osteoarthritic rabbits\u2014An in vivo pilot study","volume":"42","author":"Angrisani","year":"2021","journal-title":"Eur. Cell Mater."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1007\/978-1-0716-1425-9_10","article-title":"Multicolor Immunofluorescence Staining of Paraffin-Embedded Human Bone Marrow Sections","volume":"2308","author":"Panvini","year":"2021","journal-title":"Methods Mol. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/bs.mie.2019.05.055","article-title":"Multiplex immunohistochemistry as a novel tool for the topographic assessment of the bone marrow stem cell niche","volume":"635","author":"Bauer","year":"2020","journal-title":"Methods Enzymol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1016\/j.jos.2017.02.004","article-title":"Practical use and utility of fluorescence in situ hybridization in the pathological diagnosis of soft tissue and bone tumors","volume":"22","author":"Sugita","year":"2017","journal-title":"J. Orthop. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1007\/978-1-0716-1028-2_16","article-title":"Demineralized Murine Skeletal Histology","volume":"2230","author":"Mirando","year":"2021","journal-title":"Methods Mol. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Yakovleva, A., Plieskatt, J.L., Jensen, S., Humeida, R., Lang, J., Li, G., Bracci, P., Silver, S., and Bethony, J.M. (2017). Fit for genomic and proteomic purposes: Sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181756"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Groelz, D., Viertler, C., Pabst, D., Dettmann, N., and Zatloukal, K. (2018). Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0203608"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e27993","DOI":"10.4161\/biom.27993","article-title":"Comprehensive histological evaluation of bone implants","volume":"4","author":"Rentsch","year":"2014","journal-title":"Biomatter"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"S113","DOI":"10.1016\/j.joca.2010.05.026","article-title":"Basic methods in histopathology of joint tissues","volume":"18","author":"Schmitz","year":"2010","journal-title":"Osteoarthr. Cartil."},{"key":"ref_11","first-page":"289","article-title":"Demonstration of all connective tissue elements in a single section; pentachrome stains","volume":"60","author":"Movat","year":"1955","journal-title":"AMA Arch. Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1007\/BF02555132","article-title":"Loss of proteoglycans during decalcification of fresh metaphyses with disodium ethylenediaminetetraacetate (EDTA)","volume":"41","author":"Campo","year":"1987","journal-title":"Calcif. Tissue Int."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"545","DOI":"10.1369\/0022155419850099","article-title":"Tissue Morphology and Antigenicity in Mouse and Rat Tibia: Comparing 12 Different Decalcification Conditions","volume":"67","author":"Bogoevski","year":"2019","journal-title":"J. Histochem. Cytochem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1007\/s00441-021-03516-6","article-title":"Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections","volume":"385","author":"Nakamura","year":"2021","journal-title":"Cell Tissue Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"9844","DOI":"10.1016\/S0021-9258(18)48010-6","article-title":"Type X collagen synthesis during endochondral ossification in fracture repair","volume":"262","author":"Grant","year":"1987","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1111\/j.1601-6343.2004.00308.x","article-title":"The role of type X collagen in facilitating and regulating endochondral ossification of articular cartilage","volume":"8","author":"Shen","year":"2005","journal-title":"Orthod. Craniofac Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"12097","DOI":"10.1073\/pnas.1302703111","article-title":"Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation","volume":"111","author":"Yang","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1111\/j.1445-2197.2012.06092.x","article-title":"Formalin fixation in the \u2019-omics\u2019 era: A primer for the surgeon-scientist","volume":"82","author":"Frankel","year":"2012","journal-title":"ANZ J. Surg."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1007\/978-1-4939-8935-5_25","article-title":"An Introduction to the Performance of Immunohistochemistry","volume":"1897","author":"Magaki","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1007\/BF00306149","article-title":"The chemical and histochemical properties of Alcian Blue. I. The mechanism of Alcian Blue staining","volume":"4","author":"Scott","year":"1964","journal-title":"Histochem. Cell Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.25259\/CMAS_03_18_2021","article-title":"Nuances of the Papanicolaou stain","volume":"19","author":"Sathawane","year":"2022","journal-title":"Cytojournal"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1159\/000457827","article-title":"Inception and Development of the Papanicolaou Stain Method","volume":"61","author":"Chantziantoniou","year":"2017","journal-title":"Acta Cytol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1471","DOI":"10.1007\/s10856-007-0150-1","article-title":"Comparison of histomorphometrical data obtained with two different image analysis methods","volume":"18","author":"Ballerini","year":"2007","journal-title":"J. Mater Sci. Mater. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"666","DOI":"10.3389\/fendo.2018.00666","article-title":"An Optimized Approach to Perform Bone Histomorphometry","volume":"9","author":"Malhan","year":"2018","journal-title":"Front. Endocrinol."}],"container-title":["Bioengineering"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2306-5354\/9\/10\/488\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:36:09Z","timestamp":1760142969000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2306-5354\/9\/10\/488"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,21]]},"references-count":25,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["bioengineering9100488"],"URL":"https:\/\/doi.org\/10.3390\/bioengineering9100488","relation":{},"ISSN":["2306-5354"],"issn-type":[{"value":"2306-5354","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,21]]}}}