title: Revisiting in vivo staining with alizarin red S--a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration.
abstract: BACKGROUND
  The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals.
  
  
  RESULTS
  Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01% ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish.
  
  
  CONCLUSIONS
  The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.
authors.affiliation: Centre of Marine Sciences - CCMar, University of Algarve, Campus de Gambelas, Faro, Portugal. bensimonbrito@gmail.com.|Evolutionary Developmental Biology, Biology Department, Ghent University, Ghent, Belgium. bensimonbrito@gmail.com.|Current address: CEDOC - Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisbon, Portugal. bensimonbrito@gmail.com.
authors.forename: A
authors.initials: A
authors.lastname: Bensimon-Brito
authors.affiliation: Centre of Marine Sciences - CCMar, University of Algarve, Campus de Gambelas, Faro, Portugal. jpgsilva@ualg.pt.|ProRegeM PhD Programme, Department of Biomedical Sciences and Medicine, University of Algarve, Campus de Gambelas, Faro, Portugal. jpgsilva@ualg.pt.
authors.forename: J
authors.initials: J
authors.lastname: Cardeira
authors.affiliation: Centre of Marine Sciences - CCMar, University of Algarve, Campus de Gambelas, Faro, Portugal. gisela.dionisio@ua.pt.|Guia Marine Laboratory, Oceanography Centre, Faculty of Sciences of University of Lisbon, Cascais, Portugal. gisela.dionisio@ua.pt.
authors.forename: G
authors.initials: G
authors.lastname: Dionísio
authors.affiliation: Evolutionary Developmental Biology, Biology Department, Ghent University, Ghent, Belgium. ann.huysseune@ugent.be.
authors.forename: A
authors.initials: A
authors.lastname: Huysseune
authors.affiliation: Centre of Marine Sciences - CCMar, University of Algarve, Campus de Gambelas, Faro, Portugal. lcancela@ualg.pt.|Department of Biomedical Sciences and Medicine, University of Algarve, Campus de Gambelas, Faro, Portugal. lcancela@ualg.pt.
authors.forename: M L
authors.initials: ML
authors.lastname: Cancela
authors.affiliation: Evolutionary Developmental Biology, Biology Department, Ghent University, Ghent, Belgium. PEckhardWitten@aol.com.
authors.forename: P E
authors.initials: PE
authors.lastname: Witten
mesh_terms: D058500:Animal Fins / Q000502:physiology; D000818:Animals; D000880:Anthraquinones / Q000378:metabolism*; D001842:Bone and Bones / Q000002:abnormalities / Q000473:pathology; D002113:Calcification, Physiologic / Q000502:physiology*; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D012038:Regeneration / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation; D015027:Zebrafish / Q000254:growth & development*
chemical_list: D000880:Anthraquinones; D049452:Green Fluorescent Proteins; C004468:Alizarin Red S