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Safranin O is a cationic dye that binds to proteoglycans in cartilage and is routinely used to assess growth plate dynamics and\/or fracture repair at bone\u2013cartilage interfaces. When used with a counterstain such as fast green, safranin O can offer exquisite differentiation of cartilage from surrounding bone. However, various decalcification and processing methods can deplete proteoglycans, rendering inconsistent, weak, or absent safranin O staining with indiscriminate bone\u2013cartilage boundaries. We sought to develop an alternative staining methodology that preserves the contrast of bone and cartilage in cases of proteoglycan depletion that can be applied when other cartilage stains are unsuccessful. Here, we describe and validate a modified periodic acid\u2010Schiff (PAS) protocol that we developed using Weigert's iron hematoxylin and light green stains as an alternative to safranin O for discriminating bone\u2013cartilage interfaces of skeletal tissues. This method provides a practical solution for differentiating bone and cartilage when safranin O staining is not detected after decalcification and paraffin processing. The modified PAS protocol can be useful for studies in which identification of the bone\u2013cartilage interface is essential but may not be preserved with standard staining approaches. \u00a9 2023 The Authors.\n                    <jats:italic>JBMR Plus<\/jats:italic>\n                    published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.\n                  <\/jats:p>","DOI":"10.1002\/jbm4.10742","type":"journal-article","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T10:14:06Z","timestamp":1678875246000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Modified Periodic\n                    <scp>Acid\u2010Schiff<\/scp>\n                    (\n                    <scp>PAS<\/scp>\n                    ) Is an Alternative to Safranin O for Discriminating Bone\u2013Cartilage Interfaces"],"prefix":"10.1093","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7173-2805","authenticated-orcid":false,"given":"Kelsey M","family":"Kjosness","sequence":"first","affiliation":[{"name":"Department of Bio\u2010Medical Sciences Philadelphia College of Osteopathic Medicine  Philadelphia PA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7053-0344","authenticated-orcid":false,"given":"Philip L","family":"Reno","sequence":"additional","affiliation":[{"name":"Department of Bio\u2010Medical Sciences Philadelphia College of Osteopathic Medicine  Philadelphia PA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9211-9508","authenticated-orcid":false,"given":"Maria A","family":"Serrat","sequence":"additional","affiliation":[{"name":"Department of Biomedical Sciences, Joan C. Edwards School of Medicine Marshall University  Huntington WV USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2023,4,21]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1053\/joca.2002.0519"},{"key":"e_1_2_9_3_1","first-page":"287","volume-title":"An YH, Martin, KL, eds. Handbook of histology methods for bone and cartilage","author":"Gruber HE","year":"2003"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-416015-6.00007-1"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.2106\/00004623-197153010-00007"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1177\/22.12.1118"},{"key":"e_1_2_9_7_1","first-page":"431","volume-title":"Principles and techniques of histochemistry","author":"Troyer H","year":"1980"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00489922"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02400974"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1179\/his.2005.28.3.165"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01007021"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02555132"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02331414"},{"issue":"1","key":"e_1_2_9_14_1","first-page":"6","article-title":"After 24\u2009years in formalin, it should be fixed","author":"Skinner RA","year":"2009","journal-title":"Dent Hist"},{"key":"e_1_2_9_15_1","doi-asserted-by":"publisher","DOI":"10.1179\/his.1998.21.1.49"},{"key":"e_1_2_9_16_1","doi-asserted-by":"publisher","DOI":"10.1369\/0022155419850099"},{"key":"e_1_2_9_17_1","doi-asserted-by":"publisher","DOI":"10.1177\/34.9.2426342"},{"key":"e_1_2_9_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.matbio.2014.08.010"},{"key":"e_1_2_9_19_1","doi-asserted-by":"publisher","DOI":"10.1177\/1947603518764262"},{"key":"e_1_2_9_20_1","volume-title":"Humason's animal tissue techniques","author":"Presnell JK","year":"1997"},{"key":"e_1_2_9_21_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00279680"},{"key":"e_1_2_9_22_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0803319105"},{"key":"e_1_2_9_23_1","doi-asserted-by":"publisher","DOI":"10.1002\/ar.20480"},{"key":"e_1_2_9_24_1","doi-asserted-by":"publisher","DOI":"10.1002\/ar.a.20268"},{"key":"e_1_2_9_25_1","doi-asserted-by":"publisher","DOI":"10.1369\/0022155415591599"}],"container-title":["JBMR Plus"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jbm4.10742","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/jbm4.10742","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,3,14]],"date-time":"2024-03-14T09:58:06Z","timestamp":1710410286000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jbmrplus\/article\/7479118"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,4,21]]},"references-count":24,"journal-issue":{"issue":"6","published-print":{"date-parts":[[2023,6]]}},"alternative-id":["10.1002\/jbm4.10742"],"URL":"https:\/\/doi.org\/10.1002\/jbm4.10742","archive":["Portico"],"relation":{"has-review":[{"id-type":"doi","id":"10.1002\/JBM4.10742\/v2\/decision1","asserted-by":"object"},{"id-type":"doi","id":"10.1002\/JBM4.10742\/v2\/response1","asserted-by":"object"},{"id-type":"doi","id":"10.1002\/JBM4.10742\/v1\/review1","asserted-by":"object"},{"id-type":"doi","id":"10.1002\/JBM4.10742\/v2\/review1","asserted-by":"object"},{"id-type":"doi","id":"10.1002\/JBM4.10742\/v1\/decision1","asserted-by":"object"}]},"ISSN":["2473-4039","2473-4039"],"issn-type":[{"value":"2473-4039","type":"print"},{"value":"2473-4039","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,4,21]]},"assertion":[{"value":"2022-10-07","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-03-09","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2023-04-21","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e10742"}}