Analysis of type-Ⅰ collagen fibrils and chondroitin sulfate distribution in human dentin by confocal laser scanning microscopy combined with dual immunofluorescent labeling technique
10.3760/cma.j.issn.1002-0098.2015.12.009
- VernacularTitle:免疫荧光双重标记结合激光共聚焦扫描显微镜观察Ⅰ型胶原和硫酸软骨素在人牙本质中的分布
- Author:
Shuai LU
1
;
Sanjun ZHAO
;
Yong SUN
;
Yu GAO
;
Xiaojing LI
;
Jihua CHEN
Author Information
1. 成都军区机关医院口腔科
- Keywords:
Chondroitin sulfates;
Collagen type Ⅰ;
Dentin;
Microscopy,confocal;
Dual immunofluorescent labeling
- From:
Chinese Journal of Stomatology
2015;50(12):746-750
- CountryChina
- Language:Chinese
-
Abstract:
Objective To introduce the method of dual immunofluorescence labeling of human dentin matrix without demineralization of the whole dentin fragments, and to analyze the distribution of type-Ⅰ collagen fibrils and chondroitin sulfate in human dentin.Methods Forty 30 μm-thick middle coronal dentin sections were obtained from 8 freshly extracted human third molars and etched with 37％ phosphoric acid(PA) gel for 15 s.After preconditioning with or without tosyl-phenylalanyl chloromethyl ketone(TPCK) treated trypsin digestion, sections were subjected to dual immunofluorescent labeling and scanned by confocal laser scanning microscopy to identify the type-Ⅰ collagen fibrils and chondroitin sulfate.Results Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-Ⅰ collagen fibrils were localized in intertubular dentin and peritubular dentin.After preconditioning with TPCK treated trypsin digestion, the red fluorescence was decreased or disappeared.Conclusions The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without demineralization of the whole dentin fragments.Chondroitin sulfate was localized in the lumens of the dentin tubules and peritubular dentin, while the type-Ⅰ collagen fibrils were localized in intertubular dentin and peritubular dentin.
