Sc 365858

Immunofluorescent staining was used to quantify TNF-α, IL-10, nitrotyrosine, MOMA-2, and NF-κB p65 proteins using techniques previously described by our laboratory [86 (link)]. Tissues were stained using the following primary antibodies: TNF-α (1:250; Abcam, ab6671), IL-10 (1:200; Santa Cruz Biotechnology SC-365858), nitrotyrosine (1:200; Santa Cruz Biotechnology, SC-32757) MOMA-2 (1:500, Abcam ab33451), and NF-κB p65 (1:500, Abcam, ab86299). Secondary antibodies used were anti-rabbit Alexa Fluor 555, anti-mouse Alexa Fluor 546, anti-mouse Alexa Fluor 555, anti-rabbit Alexa Fluor Plus 488, and anti-rat Alexa Fluor 488. Slides were imaged under fluorescent microscopy at 40x with the appropriate excitation/emission filter, digitally recorded, RGB overlay signals were split and analyzed for specific fluorescence using image densitometry with Image J software (NIH). A minimum of 3–4 locations on each section (4 sections per slide), 3 slides, and n = 3 per group were used for analysis; 40x images were used for image quantification.

After sacrifice, the trimmed maxillae were fixed in 10% neutral buffered formalin for 24 h. After decalcified in ethylenediaminetetraacetic acid for 4 weeks, the tissues were then embedded by paraffin. Four-micrometer consecutive horizontal sections were obtained from the middle to apical third of the maxillary first molar, and sections from similar position of the roots were used for histological study. Immunohistochemistry was performed with a two-step detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) as previously described [39 (link)]. The positive staining cells were counted in five different slides from each sample (N = 5–6). The final result came from the average of three tests.
The primary antibodies were anti-CBS (1:100, ab135626, Abcam), anti-TNF-α (1:100, ab1793, Abcam), anti-IFN-γ (1:50, sc1377, Santa Cruz), anti-IL-10 (1:200; sc-365858, Santa Cruz, Dallas, TX), and anti-CD206 (1:200, ab64693, Abcam).

Preparation of LV homogenates, electrophoresis and western blotting were described previously [27 (link)]. The following antibodies were used: TRAF3IP2 (1:600; #bs-6202R, Bioss), p65 (1:1000; #8242, CST), phospho-p65 (Ser⁵³⁶; 1:1000; #3031, cell signaling technology, Inc or CST), c-Jun (1:1000; #9165, CST), phospho-c-Jun (Ser⁶³, 1:1000; #9261, CST), p38 MAPK (1:1000; #9212, CST), phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸², 1:1000; #9211, CST), S6K1 (1:1000, #9202, CST), phospho-S6 K (Thr³⁸⁹, 1:1000, #9205, CST), ANP (1:200, #sc20158, Santa Cruz Biotechnology), IL-10 (1:200, #sc-365858, Santa Cruz Biotechnology, Inc) and GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Inc.).