Inos antibody

L-arginine, 2-methoxyestradiol (2-Me), deoxycholic acid, hydrochloric acid, and NaOH (Wako Pure Chemicals, Osaka, Japan); N6-(1-iminoethyl)-L-lysine (L-NIL) (Cayman, Michigan, USA); cell counting kit-8 (Dojindo, Tokyo, Japan); DAF-2DA (Daiichi Pure Chemicals, Tokyo, Japan); hematoporphyrin dihydrochloride (HpD), NaCl, sodium dodecyl sulfate (SDS), Trizma base, Triton X-100, and Tween 20 (Sigma-Aldrich Japan K.K., Tokyo, Japan); NuPAGE Novex 12% Bis-Tris gels (Life Technologies Japan, Tokyo, Japan); polyvinylidene difluoride (PVDF) Blocking Reagent for Can Get Signal, Can Get Signal Immunoreaction Enhancer Solution 1, and Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo, Osaka, Japan); HIF-1α, β-actin, and horseradish peroxidase (HRP)-linked anti-rabbit IgG antibodies (Cell Signaling Technology Japan, K.K., Tokyo, Japan); HCP-1 antibody (Abcam, Cambridge, U.K.); iNOS antibody (BiossAntibodies, Massachusetts, U.S.A.) and Lumina Forte western HRP substrate (EMD Millipore, Billerica, MA, USA) were purchased and used without further purification or modification.

The fixed periodontal tissues including alveolar bones were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 5 weeks and embedded in paraffin and cut by 5 μm. The sections were stained with hematoxylin and eosin. For the analyses of inflammatory cell infiltration, the sections were incubated with anti-inducible nitric oxide synthase (iNOS) antibody (Bioss, Boston, USA). Although iNOS is expressed mainly in inflammatory cells, other type of cells such as vascular smooth muscle cells express iNOS in some cases. Therefore, we calculated the number of iNOS-positive rounded cells as iNOS-positive inflammatory cells under light microscopy (×400) by one skilled investigator.
To observe the osteoclasts and osteoblasts, the sections were stained with tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) (Sept. Sapie Co., Ltd., Tokyo, Japan). Osteoclasts were defined as multinucleated, TRAP-positive cells in contact with the surface of the alveolar bone [18 (link)]. The activities of osteoblasts were evaluated by the intensity of ALP using ImageJ. The number of TRAP-positive cells and the intensity of ALP per the length of alveolar bone surface were calculated.