Antibodies against hif 1α

Rat periodontal tissues were fixed in 4% PFA for 48 h and decalcified with 12% ethylenediaminetetraacetic acid (EDTA, pH 5.6) at room temperature for 21 days. The sections were paraffin embedded and cut serially in the sagittal plane from the most lingual side. Semi-serial 5-μm sections of first molars were prepared and stained according to immunohistochemistry protocols. After antigen retrieval by heat-induced epitope retrieval, deparaffinized sections were immersed in 0.6% H₂O₂ for 20 min to quench endogenous peroxidase activity. Sections were then blocked in 5% BSA for 30 min and incubated with antibodies against HIF-1α (0.05 μg/ml), VEGF (0.05 μg/ml), or RUNX2 (0.05 μg/ml) (all from Abcam) overnight at 4°C. Sections were then incubated with goat anti-rabbit or mouse IgG antibodies for 1 h at room temperature and reacted with avidin-biotin-peroxidase complexes (Vector Laboratories, USA) in PBS for 30 min. After colour development with 0.05% 3,3′-diaminobenzidine, sections were counterstained with haematoxylin.

HUVECs were cultured in ECM containing 1% FBS and treated with PPD (2.5 μM) for the indicated time periods. Soluble chromatin including the VEGF promoter region was isolated using the SimpleChIP plus enzymatic chromatin IP kit (Cell Signaling Technology), as described in the manufacturer’s manual. Briefly, the cells were fixed with formaldehyde and lysed. Chromatin was harvested and subjected to immunoprecipitation using antibodies against HIF-1α (Abcam). The DNA in the co-precipitated chromatin was extracted and used as a template for real-time PCR. The reaction was performed to amplify the VEGF promoter. The Ct value for HIF-1α antibody captured DNA was normalized to that of the input DNA. Data were presented as fold enrichment over input DNA.