Gpx 1

Levels of protein expression of hippocampal tissue (including whole tissue cell lysate, cytosolic or nuclear fractions) were carried out as previously described [23 (link)]. The optical density of the bands was measured and quantified by Image J (National Institute of Health, MD, USA). Primary antibodies of SOD-2, GPx-1, NFκB p65 and p50, IκBα, TNFα, IL-1β, IL-6 and COX-2 were purchased from Santa Cruz Biotechnology, CA, USA; Synapsin-1 and Synaptophysin were purchased from Novus Biologicals, USA; PSD95 and Cleaved Caspase 3 was purchased from Cell Signaling Technology; Cleaved PARP1 was purchased from Bioworld Technology; IDO-1 was purchased from antibodies-online (ABIN1714836). The data were expressed as percentage of the control.

Maxillae tissue was deparaffinized and rehydrated. Gingival and periodontal tissue slices (4 μm) were washed with 0.3% Triton X-100 in phosphate buffer, quenched with endogenous peroxidase (3% hydrogen peroxide), and incubated overnight at 4°C with primary antibodies against the following proteins (all antibodies 1:400): receptor activator of the NF-κB ligand (RANKL), superoxide dismutase-1 (SOD-1), receptor activator of NF-κB (RANK), glutathione peroxidase-1 (GPx-1), osteoprotegerin (OPG), matrix metalloproteinase 2 (MMP-2), cathepsin K, and cyclooxygenase-2 (Santa Cruz Biotechnology, INTERPRISE, São Paulo, SP, Brazil) and incubated for 30 min with a streptavidin/horseradish peroxidase-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). Immunostaining was visualized using colorimetric detection (Biocare Medical, Dakota, USA). The staining status was identified as either negative or positive; positive staining was defined as the presence of brown chromogen. Staining intensity and the proportion of immunopositive cells were examined independently by two pathologists by light microscopy and recorded. Intensity of staining (IS) was graded on a 0 to 2 scale according to the following semi-quantitative assessment: 0 = no detectable staining, 1 = weak staining; 2 = strong staining.²²

Protein expressions of hippocampal tissue lysate (including cytosolic and nuclear fractions) were performed as previously described [26 (link)]. The expression of β-actin served as the internal control for whole cell lysate and cytosolic fraction protein, whereas Lamin B1 served as the internal control of nuclear fraction protein. Primary antibodies of SOD-2 (rabbit polyclonal, 1:1000), GPx-1 (goat polyclonal, 1:500), NFκB p65 (rabbit polyclonal, 1:250) and p50 (mouse monoclonal, 1:250), IκBα (mouse monoclonal, 1:500), TNF α (goat polyclonal, 1:80), IL-1β (rabbit polyclonal 1:100), IL-6 (goat polyclonal, 1:1000) and COX-2 (goat polyclonal, 1:100) were purchased from Santa Cruz Biotechnology, CA, USA; Synapsin 1 (rabbit polyclonal, 1:500) and Synaptophysin (rabbit polyclonal, 1:2000) were purchased from Novus Biologicals, USA; PSD95 (rabbit polyclonal 1:500), Cleaved Caspase 3 (rabbit polyclonal 1:500) was purchased from Cell Signaling Technology; Cleaved PARP-1 (rabbit polyclonal, 1:2000) was purchased from Bioworld Technology; IDO-1 (rabbit polyclonal, 1:250) was purchased from antibodies-online (ABIN1714836). The optical density of the bands was measured and quantified by Image J (National Institute of Health, MD, USA). The data were expressed as percentage of the control.