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Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

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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 mu CT 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.
Keywords
Fluorescence imaging, Calcium, Bone, Mineral apposition, Caudal fin, Vertebral column, Hydroxyapatite, Alizarin red S, SALMON SALMO-SALAR, ATLANTIC SALMON, DANIO-RERIO, BONE REGENERATION, TELEOST FISH, FIN, MARKING, GROWTH, MODEL, OSTEOBLASTS

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Chicago
Bensimon-Brito, A, J Cardeira, G Dionísio, Ann Huysseune, ML Cancela, and Paul Witten. 2016. “Revisiting in Vivo Staining with Alizarin Red S - a Valuable Approach to Analyse Zebrafish Skeletal Mineralization During Development and Regeneration.” Bmc Developmental Biology 16.
APA
Bensimon-Brito, A, Cardeira, J., Dionísio, G., Huysseune, A., Cancela, M., & Witten, P. (2016). Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration. BMC DEVELOPMENTAL BIOLOGY, 16.
Vancouver
1.
Bensimon-Brito A, Cardeira J, Dionísio G, Huysseune A, Cancela M, Witten P. Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration. BMC DEVELOPMENTAL BIOLOGY. 2016;16.
MLA
Bensimon-Brito, A, J Cardeira, G Dionísio, et al. “Revisiting in Vivo Staining with Alizarin Red S - a Valuable Approach to Analyse Zebrafish Skeletal Mineralization During Development and Regeneration.” BMC DEVELOPMENTAL BIOLOGY 16 (2016): n. pag. Print.
@article{7223711,
  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 mu CT 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.},
  articleno    = {2},
  author       = {Bensimon-Brito, A and Cardeira, J and Dion{\'i}sio, G and Huysseune, Ann and Cancela, ML and Witten, Paul},
  issn         = {1471-213X},
  journal      = {BMC DEVELOPMENTAL BIOLOGY},
  language     = {eng},
  pages        = {9},
  title        = {Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration},
  url          = {http://dx.doi.org/10.1186/s12861-016-0102-4},
  volume       = {16},
  year         = {2016},
}

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