A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system
A 50% (w/v, equivalent to 2.943 mol/L) silver nitrate solution is commonly used to stain and characterize the osteocyte lacuno-canalicular system (LCS) in bone biology research. However, variability in reagent concentrations and types, along with inconsistent staining procedures, have limited the br...
Saved in:
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2025-05-01
|
| Series: | Frontiers in Endocrinology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fendo.2025.1561576/full |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849321565169123328 |
|---|---|
| author | Jinlian Wu Jinlian Wu Chunchun Xue Qiang Li Hongjin Wu Jie Zhang Chenglong Wang Weiwei Dai Libo Wang |
| author_facet | Jinlian Wu Jinlian Wu Chunchun Xue Qiang Li Hongjin Wu Jie Zhang Chenglong Wang Weiwei Dai Libo Wang |
| author_sort | Jinlian Wu |
| collection | DOAJ |
| description | A 50% (w/v, equivalent to 2.943 mol/L) silver nitrate solution is commonly used to stain and characterize the osteocyte lacuno-canalicular system (LCS) in bone biology research. However, variability in reagent concentrations and types, along with inconsistent staining procedures, have limited the broader application of this method in osteocyte research. In this study, we present a novel optimized silver nitrate staining technique aimed at addressing these limitations. This new method utilizes a 1 mol/L (equivalent to 16.987%, w/v) silver nitrate solution in combination with a type-B gelatin-formic acid solution at various concentrations (0.05%-0.5% gelatin and 0.05%-5% formic acid, or 1%-2% gelatin and 0.1%-2% formic acid) in volume ratios of 4:1, 2:1, or 1:1, or a 0.5 mol/L silver nitrate solution at a 4:1 ratio. The staining process is carried out for 1 hour under ultraviolet light or 90 minutes under regular room light (or dark), followed by washing with Milli-Q water to terminate the reaction. We applied this new method to stain the osteocyte LCS in bone samples from different species and pathological bone models. The technique consistently produced clear, distinct staining patterns across all samples. Moreover, our novel method revealed a greater number of LCS compared to the traditional 50% silver nitrate solution. This suggests that the commonly used 50% silver nitrate method may disrupt or inadequately reveal the LCS in bones, potentially leading to an underestimation of LCS density and number. In conclusion, our novel silver nitrate staining method provides a simpler and more cost-effective alternative to the traditional technique. By offering a more accurate and comprehensive analysis of the LCS across species, this approach has the potential to advance research on osteocyte morphogenesis, as well as the functional and evolutionary adaptations of the osteocyte LCS across different taxa. |
| format | Article |
| id | doaj-art-8f7f0ed36ca84302a569bc00f4c57f32 |
| institution | Kabale University |
| issn | 1664-2392 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Endocrinology |
| spelling | doaj-art-8f7f0ed36ca84302a569bc00f4c57f322025-08-20T03:49:42ZengFrontiers Media S.A.Frontiers in Endocrinology1664-23922025-05-011610.3389/fendo.2025.15615761561576A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular systemJinlian Wu0Jinlian Wu1Chunchun Xue2Qiang Li3Hongjin Wu4Jie Zhang5Chenglong Wang6Weiwei Dai7Libo Wang8Central Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaDepartment of Rheumatology and Immunology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaShanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaLonghua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaCentral Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaCentral Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaCentral Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaCentral Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaCentral Laboratory of Science and Technology, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai, ChinaA 50% (w/v, equivalent to 2.943 mol/L) silver nitrate solution is commonly used to stain and characterize the osteocyte lacuno-canalicular system (LCS) in bone biology research. However, variability in reagent concentrations and types, along with inconsistent staining procedures, have limited the broader application of this method in osteocyte research. In this study, we present a novel optimized silver nitrate staining technique aimed at addressing these limitations. This new method utilizes a 1 mol/L (equivalent to 16.987%, w/v) silver nitrate solution in combination with a type-B gelatin-formic acid solution at various concentrations (0.05%-0.5% gelatin and 0.05%-5% formic acid, or 1%-2% gelatin and 0.1%-2% formic acid) in volume ratios of 4:1, 2:1, or 1:1, or a 0.5 mol/L silver nitrate solution at a 4:1 ratio. The staining process is carried out for 1 hour under ultraviolet light or 90 minutes under regular room light (or dark), followed by washing with Milli-Q water to terminate the reaction. We applied this new method to stain the osteocyte LCS in bone samples from different species and pathological bone models. The technique consistently produced clear, distinct staining patterns across all samples. Moreover, our novel method revealed a greater number of LCS compared to the traditional 50% silver nitrate solution. This suggests that the commonly used 50% silver nitrate method may disrupt or inadequately reveal the LCS in bones, potentially leading to an underestimation of LCS density and number. In conclusion, our novel silver nitrate staining method provides a simpler and more cost-effective alternative to the traditional technique. By offering a more accurate and comprehensive analysis of the LCS across species, this approach has the potential to advance research on osteocyte morphogenesis, as well as the functional and evolutionary adaptations of the osteocyte LCS across different taxa.https://www.frontiersin.org/articles/10.3389/fendo.2025.1561576/fullsilver nitrategelatinformic acidosteocytelacuno-canalicular system (LCS) |
| spellingShingle | Jinlian Wu Jinlian Wu Chunchun Xue Qiang Li Hongjin Wu Jie Zhang Chenglong Wang Weiwei Dai Libo Wang A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system Frontiers in Endocrinology silver nitrate gelatin formic acid osteocyte lacuno-canalicular system (LCS) |
| title | A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system |
| title_full | A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system |
| title_fullStr | A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system |
| title_full_unstemmed | A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system |
| title_short | A novel optimized silver nitrate staining method for visualizing the osteocyte lacuno-canalicular system |
| title_sort | novel optimized silver nitrate staining method for visualizing the osteocyte lacuno canalicular system |
| topic | silver nitrate gelatin formic acid osteocyte lacuno-canalicular system (LCS) |
| url | https://www.frontiersin.org/articles/10.3389/fendo.2025.1561576/full |
| work_keys_str_mv | AT jinlianwu anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT jinlianwu anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT chunchunxue anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT qiangli anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT hongjinwu anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT jiezhang anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT chenglongwang anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT weiweidai anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT libowang anoveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT jinlianwu noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT jinlianwu noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT chunchunxue noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT qiangli noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT hongjinwu noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT jiezhang noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT chenglongwang noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT weiweidai noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem AT libowang noveloptimizedsilvernitratestainingmethodforvisualizingtheosteocytelacunocanalicularsystem |