Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis
Abstract Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. T...
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2025-01-01
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| author | Shibarjun Mandal Ramya Motganhalli Ravikumar Astrid Tannert Annett Urbanek Rustam R. Guliev Max Naumann Sina M. Coldewey Uta Dahmen Lina Carvalho Luís Bastião Silva Ute Neugebauer |
| author_facet | Shibarjun Mandal Ramya Motganhalli Ravikumar Astrid Tannert Annett Urbanek Rustam R. Guliev Max Naumann Sina M. Coldewey Uta Dahmen Lina Carvalho Luís Bastião Silva Ute Neugebauer |
| author_sort | Shibarjun Mandal |
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| description | Abstract Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2–3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements. |
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| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-5bcee28d7e684d11997b4bc2248fb6092025-01-12T12:24:31ZengNature PortfolioScientific Reports2045-23222025-01-0115111510.1038/s41598-024-84330-2Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysisShibarjun Mandal0Ramya Motganhalli Ravikumar1Astrid Tannert2Annett Urbanek3Rustam R. Guliev4Max Naumann5Sina M. Coldewey6Uta Dahmen7Lina Carvalho8Luís Bastião Silva9Ute Neugebauer10Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Leibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Center for Sepsis Control and Care, Jena University HospitalExperimental Surgery, Clinic for General, Visceral and Vascular Surgery, Jena University HospitalInstitute of Anatomical and Molecular Pathology, Faculty of Medicine, University of CoimbraBMD SoftwareLeibniz Institute of Photonic Technology (Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI)Abstract Bone tissue, with its complex structure, often necessitates decalcification of the hard tissue for ex vivo morphological studies. The choice of a suitable decalcification method plays a crucial role in preserving desired features and ensuring compatibility with diverse imaging techniques. The search for a universal decalcification method that is suitable for a range of biophotonic analyses remains an ongoing challenge. In this study, we systematically assessed five standard bone decalcification protocols, encompassing strong mineralic acids (3% and 5% nitric acid), a commercially available formulation of hydrochloric and formic acid), as well as weak organic acids (5% trichloroacetic acid and 8% formic acid), and a chelating agent (25% ethylenediamine-tetraacetic acid) with varying decalcification durations, using mouse long bones as our experimental model. Our imaging analysis panel included classical histological staining (Hematoxylin and Eosin, H&E), immunofluorescence staining, and label-free Raman microspectroscopic imaging. We used cryosections instead of paraffin sections since paraffin interferes with tissue Raman signals. This approach is not as commonly used as it is more prone to handling artifacts, but is the preferred method for subsequent Raman analysis. Decalcification efficacy was evaluated based on various qualitative and some quantitative imaging parameters by 2–3 independent observers. Our systematic approach revealed that the chelating agent, when used for 24 h, optimally preserved bone features and, thus, would be the ideal decalcifying agent for comprehensive subsequent analysis. However, the choice of decalcifier and the ideal decalcification duration may vary depending on the type and thickness of bone, necessitating tailored adjustments to meet specific experimental requirements.https://doi.org/10.1038/s41598-024-84330-2DecalcificationRaman imagingBoneHistology stainingImmunofluorescence labellingConfocal laser scanning microscopy |
| spellingShingle | Shibarjun Mandal Ramya Motganhalli Ravikumar Astrid Tannert Annett Urbanek Rustam R. Guliev Max Naumann Sina M. Coldewey Uta Dahmen Lina Carvalho Luís Bastião Silva Ute Neugebauer Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis Scientific Reports Decalcification Raman imaging Bone Histology staining Immunofluorescence labelling Confocal laser scanning microscopy |
| title | Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| title_full | Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| title_fullStr | Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| title_full_unstemmed | Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| title_short | Qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| title_sort | qualitative comparison of decalcifiers for mouse bone cryosections for subsequent biophotonic analysis |
| topic | Decalcification Raman imaging Bone Histology staining Immunofluorescence labelling Confocal laser scanning microscopy |
| url | https://doi.org/10.1038/s41598-024-84330-2 |
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