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Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.<\/jats:p>","DOI":"10.3390\/biom13121780","type":"journal-article","created":{"date-parts":[[2023,12,12]],"date-time":"2023-12-12T04:28:31Z","timestamp":1702355311000},"page":"1780","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection"],"prefix":"10.3390","volume":"13","author":[{"given":"Ann","family":"Huysseune","sequence":"first","affiliation":[{"name":"Research Group Evolutionary Developmental Biology, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium"},{"name":"Department of Zoology, Faculty of Science, Charles University, Vinicna 7, 128 44 Prague, Czech Republic"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ulrike G.","family":"Larsen","sequence":"additional","affiliation":[{"name":"Department for Biomedicine, Aarhus University, H\u00f8egh-Guldbergs Gade 10, 8000 Aarhus, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1700-3569","authenticated-orcid":false,"given":"Daria","family":"Larionova","sequence":"additional","affiliation":[{"name":"Research Group Evolutionary Developmental Biology, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3062-3896","authenticated-orcid":false,"given":"Cecilie L.","family":"Matthiesen","sequence":"additional","affiliation":[{"name":"Department for Biomedicine, Aarhus University, H\u00f8egh-Guldbergs Gade 10, 8000 Aarhus, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9338-911X","authenticated-orcid":false,"given":"Steen V.","family":"Petersen","sequence":"additional","affiliation":[{"name":"Department for Biomedicine, Aarhus University, H\u00f8egh-Guldbergs Gade 10, 8000 Aarhus, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6030-2164","authenticated-orcid":false,"given":"Marc","family":"Muller","sequence":"additional","affiliation":[{"name":"Laboratoire d\u2019Organogen\u00e8se et R\u00e9g\u00e9n\u00e9ration, GIGA-R 1, Avenue de l\u2019H\u00f4pital, B34 Sart Tilman, 4000 Li\u00e8ge, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2928-5762","authenticated-orcid":false,"given":"P. Eckhard","family":"Witten","sequence":"additional","affiliation":[{"name":"Research Group Evolutionary Developmental Biology, Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2853","DOI":"10.1016\/j.cell.2022.06.010","article-title":"Nitric oxide signaling in health and disease","volume":"185","author":"Lundberg","year":"2022","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1881","DOI":"10.1152\/physrev.00005.2022","article-title":"The superoxide radical switch in the biology of nitric oxide and peroxynitrite","volume":"102","author":"Piacenza","year":"2022","journal-title":"Physiol. 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