Sc 32757

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.

Mouse brains were rapidly dissected on ice and hippocampus tissues were homogenized in a lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% SDS and protease inhibitors (Complete Mini; Roche, Basel, Switzerland). After centrifugation at 4 °C (14,000 rpm for 10 min), cellular debris was removed, and the supernatant was collected for western blotting. Tissue lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and separated proteins were transferred to nitrocellulose membranes. Membranes were then blocked with 5% defatted milk in Tris-buffered saline with Tween 20 (TBST) for 1 h and incubated overnight at 4 °C with the following specific primary antibodies against iNos (Abcam, ab178945; dilution 1:1000), nitrotyrosine (Santa Cruz: sc-32757; dilution 1:1000) and Hsp60 (Abcam, ab46798; dilution 1:2000). Anti-GAPDH antibody (AF0006, Beyotime, Jiangsu, China) was used as a loading control. After three washes with TBST, an HRP-labeled secondary antibody (CWS, Taizhou, China) was added at room temperature for 1 h using 5% milk in TBST, followed by three additional washes with TBST. The Immobilon ECL western system (Millipore, Burlington, USA) was then used to visualize the bands, which were quantified and analyzed with Gel-Pro Analysis software (Media Cybernetics, Rockville, MD, USA).