Technical advances in the sectioning of dental tissue and of on-section cross-linked collagen detection in mineralized teeth.
Immunohistochemical detection of cross-linked fibrillar collagens in mineralized tissues is much desired for exploring the mechanisms of biomineralization in health and disease. Mineralized teeth are impossible to section when embedded in conventional media, thus limiting on-section characterization of matrix proteins by immunohistochemistry. We hypothesized that by using an especially formulated acrylic resin suitable for mineralized dental tissues, not only sectioning of teeth would be possible, but also our recently developed immunofluorescence labeling technique would be amenable to fully calcified tissues for characterization of dentinal fibrillar collagens, which remains elusive. The hypothesis was tested on fixed rodent teeth embedded in Technovit 9100 New. It was possible to cut thin (1 mum) sections of mineralized teeth, and immunofluorescence characterization of cross-linked type I fibrillar collagen was selected due to its abundance in dentine. Decalcified samples of teeth embedded in paraffin wax were also used to compare immunolabeling from either method using the same immunoreagents in equivalent concentrations. In the decalcified tissue sections, type I collagen labeling in the dentine along the tubules was "patchy" and the signal in the predentine was very weak. However, enhanced signal in mineralized samples with type I collagen was detected not only in the predentine but also at the limit between intertubular dentine, within the elements of the enamel organ and subgingival stroma. This report offers advances in sectioning mineralized dental tissues and allows the application of immunofluorescence not only for on-section protein detection but importantly for detecting cross-linked fibrous collagens in both soft and mineralized tissue sections.
Singhrao
Sim K
SK
Connective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, Cardiff, CF10 3US, Wales, UK. Singhrao@cf.ac.uk
Sloan
Alastair J
AJ
Smith
Emma L
EL
Archer
Charles W
CW
D000180:Acrylic Resins; D000818:Animals; D024042:Collagen Type I / Q000032:analysis; D003804:Dentin / Q000737:chemistry / Q000648:ultrastructure; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D016591:Histocytological Preparation Techniques / Q000379:methods*; D007150:Immunohistochemistry; D008297:Male; D008853:Microscopy; D016612:Paraffin Embedding; D051381:Rats; D017208:Rats, Wistar; D014070:Tooth / Q000648:ultrastructure*; D014074:Tooth Calcification
D000180:Acrylic Resins; D024042:Collagen Type I; D005404:Fixatives