Ab9969

Mouse brain tissue and colon tissue were collected from each group. Protein samples were separated on SDS−PAGE gels then electrotransferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). The primary antibodies were hybridized with membranes. The primary antibodies included anti-IL-10 (1:200, ab9969, Abcam, Cambridge, MA, USA) and anti-IL-17 (1:200, ab79056, Abcam, Cambridge, MA, USA). The secondary antibodies incubated with membranes. The ImageJ software were used to scan and analyze protein bands. The bands of brain tissue were normalized to β-tubulin (ab 7291, Abcam, Cambridge, MA, USA), and colon tissue were normalized to β-actin (ab8227, Abcam, Cambridge, MA, USA).

After dewaxing and hydration of the alveolar bone section, antigen retrieval was done in boiling Tris-EDTA solution (pH = 9.0) for 15 to 20 minutes. Endogenous peroxidase was blocked by 3% hydrogen peroxide, followed by incubation with 10% normal goat serum. Incubation with primary antibodies (Abcam, Cambridge, MA) anti-CD68 (1:100, ab31630), tumor necrosis factor-α (TNF-α) (1:100, ab6671), interleukin (IL)-1β (10 μg/mL, ab9722), or IL-10 (1:100, ab9969) was done, respectively, for 2 or 3 hours at 37 centigrade. Incubation with a ready-to-use secondary antibody coupled with horseradish peroxidase (goat-anti-rabbit) (Polink-1, Golden Bridge International, Mukilteo, WA) was done for 30 minutes at 37 centigrade. For color reaction, 3,3′-diaminobenzidine was used. Phosphate buffered saline was used for blank control. The tissue was counterstained with hematoxylin. CD68⁺ cells (i.e., macrophages) around the second molar were counted and calculated the same as leukocyte counting. Protein levels were quantified as integrated optical density/area of interest (i.e., mean optical density) using image analysis software (Image Pro Plus, Media Cybernetics, Silver Spring, MD). Area of interest was restricted to the suprabony soft tissue mesial or distal to the second molar (200×). For each target protein, three discontinuous sections were stained for analysis.

Tissue homogenates from the PFC and hippocampus were subjected to Western Blot (WB) analysis (n = 6/group) for the determination of protein levels of IL-1β, IL-4, IL-10, TLR4, and β-Actin. The extraction of protein and WB was performed as previously described (Agarwal et al., 2011 (link)). Primary antibodies were commercially obtained: IL-1β, and β-Actin, 1:1000 dilution (SC-7884 and SC-1616R respectively, Santa Cruz Biotechnology, Santa Cruz, CA), TLR4, IL-4, and IL-10, 1:1000 dilution (ab13556, ab9811, and ab9969 respectively, Abcam, Cambridge, MA). Secondary antibodies were commercially obtained: anti-rabbit, 1:5000 dilution (SC-2004, Santa Cruz Biotechnology, Santa Cruz, CA).

Hippocampal TNF-α and IL-10 levels were measured via western blotting. Western blotting was performed as mentioned above, but without a blocking step. Primary antibodies for TNF-α (AB1837P; 1:2500, i.e., 0.2 μg/mL, Merck Millipore, Darmstadt, Germany) and for IL-10 (ab9969; 1:10000 dilution; i.e., 0.1 μg/mL; Abcam, Cambridge, MA, USA), and secondary antibody for rabbit (ab16284 1:10000 = 0.1 μg/mL, Abcam, Cambridge, MA, USA) were used.