Il 1β primary antibody

Collected media were concentrated (approximately 20×) using a centrifugal concentrator with a molecular weight cut-off of 3 kDa (Millpore Merck, Darmstadt, German). Aliquots of collected cells and concentrated medium were used for total protein extraction using RIPA buffer and then for Western blot analysis, as described previously [12 (link),14 (link)]. A rabbit anti-chicken IL-1β primary antibody (Abcam, Cambridge, UK) and antibodies cross-reactive to chicken antigens, including rabbit anti-VDR (vitamin D receptor, Cat.# GR37-100UGCN, Merck Billerica, MA, USA), anti-p65 (a subunit of NFκB, nuclear factor kappa B, Cat.# 8242), and β-actin (Cat.# 4967, Cell Signaling Technology, Beverly, MA, USA), and a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Beverly, MA, USA) were used in Western blot study.

After scanning, the specimens were decalcified, dewatered, and embedded in paraffin. Then, they were mounted in the medial plane on 4-μm serial sections on anti-adhesive slides in preparation for HE staining, Masson staining, as well as immunohistochemical (IHC) staining. IL-1β primary antibody (dilution 1:1000, Abcam, USA) and TGF-β primary antibody (dilution 1:1000, Abcam, USA) was used for IHC staining. Three consecutive square fields involving junctional epithelium (JE), the gingival epithelium (GE), and alveolar bone (AB) in the mesial coronal area of the first molar were selected from each section. Six sections of each group were analyzed, and each measurement was counted by the examiner in a blind manner.