IADR Abstract Archives
Effect of Decalcification Methods on Mouse Periodontal Tissue Histological Assessment
Objectives: Decalcification is an important procedure performed for the histopathological observation of mineralized tissues like bone and teeth. Selection of the decalcification agents must be carefully considered when processing mineralized tissues since the integrity and immunohistochemical characteristics of the samples may be affected. In a recent paper, Tainaka et. al. (Cell Rep. 2018) reported that the combination of EDTA and imidazole accelerate the decalcification process without affecting the luminance of the fluorescence proteins. However, it remains elusive whether after the decalcification procedure the tissue still retains its morphological characteristics and immunoreactivity. This study aimed to evaluate the suitability of a new decalcifying reagent and condition for histological assessment in mouse periodontal tissue.
Methods: Two decalcifying agents (EDTA/NaOH (EN) and EDTA/Imidazole (EI)) and 2 temperature conditions (Room temperature (RT) and 37oC) were evaluated for the decalcification of the paraformaldehyde-fixed mice mandibles (C57BL/6, 12-week old). The decalcification rate was evaluated by using micro-CT scan images (Rigaku) and analyzed with Analyze 12.0 software (AnalyzeDirect). Morphological preservation of the samples was observed with Hematoxylin-Eosin staining (HE) on paraffin-embedded histology section. Preservation of the immunoreactivity was evaluated with immunofluorescence staining.
Results: Complete decalcification of 12-week old mice mandibles at RT was achieved at 7 days by EN and 6 days by EI. Decalcification of the samples at 37oC showed a significant shortening of the decalcification time to 4 days for EN and 2 days for EI. HE staining of EI samples showed no apparent morphological changes when compared with EN. Immunoreactivity against selected antibodies was retained in all the conditions used in this study.
Conclusions: A new decalcifying reagent at 37oC showed a significantly shorter decalcification time without affecting the histological morphology and immunoreactivity against selected antibodies, suggesting as an optimal decalcification solution for the periodontal tissue histological assessment.
Methods: Two decalcifying agents (EDTA/NaOH (EN) and EDTA/Imidazole (EI)) and 2 temperature conditions (Room temperature (RT) and 37oC) were evaluated for the decalcification of the paraformaldehyde-fixed mice mandibles (C57BL/6, 12-week old). The decalcification rate was evaluated by using micro-CT scan images (Rigaku) and analyzed with Analyze 12.0 software (AnalyzeDirect). Morphological preservation of the samples was observed with Hematoxylin-Eosin staining (HE) on paraffin-embedded histology section. Preservation of the immunoreactivity was evaluated with immunofluorescence staining.
Results: Complete decalcification of 12-week old mice mandibles at RT was achieved at 7 days by EN and 6 days by EI. Decalcification of the samples at 37oC showed a significant shortening of the decalcification time to 4 days for EN and 2 days for EI. HE staining of EI samples showed no apparent morphological changes when compared with EN. Immunoreactivity against selected antibodies was retained in all the conditions used in this study.
Conclusions: A new decalcifying reagent at 37oC showed a significantly shorter decalcification time without affecting the histological morphology and immunoreactivity against selected antibodies, suggesting as an optimal decalcification solution for the periodontal tissue histological assessment.