Posted at the Institutional Resources for Unique Collection and Academic Archives at Tokyo Dental College, Available from http://ir.tdc.ac.jp/
Title
Combined effects of systemic parathyroid hormone (1‑34) and locally delivered neutral self‑
assembling peptide hydrogel in the treatment of periodontal defects: An experimental in vivo investigation
Author(s) Alternative
Yoshida, W; Matsugami, D; Murakami, T; Bizenjima, T; Imamura, K; Seshima, F; Saito, A
Journal Journal of clinical periodontology, 46(10): 1030‑
1040
URL http://hdl.handle.net/10130/5214
Right
This is the peer reviewed version of the following article: J Clin Periodontol. 2019 Oct;46(10):1030‑
1040., which has been published in final form at https://doi.org/10.1111/jcpe.13170. This article may be used for non‑commercial purposes in
accordance with Wiley Terms and Conditions for Use of Self‑Archived Versions.
Description
Yoshida et al.
Supplemental file; Appendix S3.
Detailed methods for Immunohistochemistry
Immunohistochemistry was performed essentially as described by Bizenjima et al.
(2015) and Takeuchi et al. (2016). Paraffin sections were deparaffinized with xylol and incubated in 3% hydrogen peroxide with methanol for 30 min. at room temperature. For antigen retrieval, the sections of all antibodies were treated with microwave oven for 3 min. After washing with phosphate-buffered saline (PBS, pH 7.4), sections were
incubated with 3% bovine serum albumin for 30 min. to block non-specific binding. For the analysis of proliferating cell nuclear antigen (PCNA), the sections were incubated for 12 h at 4°C with mouse anti-PCNA primary antibody (PC-10, DAKO, Carpinteria, CA, USA) at a dilution of 1:100. Then, sections were incubated with a biotinylated secondary antibody [Histofine Simple Stain Max PO (M), Nichirei, Tokyo] for 60 min.
at room temperature. Thereafter, they were rinsed with PBS and stained with diaminobenzidine (Histofine Simple Stain DAB, Nichirei) and counterstained with haematoxylin. A brown coloration indicated a PCNA-positive reaction.
For the detection of vascular endothelial growth factor (VEGF) expression, anti- VEGF monoclonal antibodies (dil.1:50; Abcam, Tokyo) were used. To confirm
specificity of staining, a non-immune mouse IgG (Abcam) control was used at the same concentration.
To detect osterix (Osx), an anti-Osx polyclonal antibody (1:500; Abcam) was used.
The sections were subsequently incubated with a biotinylated secondary antibody (Histofine® Simple Stain Max PO (R) , Nichirei) for 60 min. at room temperature.
[Quantitative analysis]
PCNA-positive or VEGF-positive cells were counted in each of the three
compartmentalized regions (Root side, Bone side, and Middle area) under a light microscope at × 200 magnification. Osx-positive cells were counted in two regions (Root side and Bone side). A 350 × 470 μm area, randomly selected within the
compartmentalized region in each section, was submitted to quantitative analysis. The analysis was performed using an image analyzer (Image-Pro Plus 6.2; Media
Cybernetics division of Nippon Roper, Tokyo, Japan), as described by Takeuchi et al.
(2016). Cell counting was performed by one examiner and confirmed by a second independent examiner.
