Rabbit anti iba 1

Four series of sections were deparaffinized in xylene and rehydrated. Endogenous peroxidase activity was extinguished with 10% H₂O₂ and heat-induced antigen retrieval was performed using Tris-EDTA buffer (1 mM EDTA solution, 10 mM Tris base, and 0.05% Tween 20 in distilled water, pH 9). Sections were incubated overnight in a buffer solution (5% skim milk, 0.1% Tween 20 in Tris-buffered saline) containing one of the following primary antibodies: rabbit anti-TNF-α, rabbit anti-iba1, goat anti-caspase 3, and goat anti-Fas (1 : 100, Santa Cruz Biotechnology, CA). Then, sections were incubated in an HRP-conjugated secondary antibody and processed according to the instructions of the kit manufacturer (ABCAM, Cambridge, UK). Finally, they were stained with chromogen substrate 3,3′-diaminobenzidine hydrochloride (DAB, ABCAM) (10 min), counterstained with hematoxylin (ABCAM) (5 min), dehydrated through a graded ethanol series, cleared in xylene, and covered with a glass coverslip. Tris-TBS buffer (0.1% Tween 20 in Tris-buffered saline, pH = 7.6) was used for interstep rinsing, as recommended by the kit manufacturer (ABCAM). Expressions of these markers of inflammation (Tumor Necrosis Factor-alpha/TNF-α, iba1), inflammation-related cell death (Fas receptor), and apoptosis (caspase-3) were observed using a light microscope under 20x, 40x, and 120x objectives.

Ten percent SDS-PAGE was used to separate protein samples (about 80 μg protein), which were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). PVDF membranes were then blocked by 5% BSA for 1 h at 37°C. PVDF membranes were cut off in accordance with the molecular weight, and incubated with different primary antibodies (rabbit anti-GAPDH, 1:1000 dilution; mouse anti-RAGE, 1:800; rabbit anti-p-NF-κB P65, 1:800; mouse anti-GFAP, 1:1000; rabbit anti-Iba-1, 1:800; mouse anti-p-JNK, 1:800; rabbit anti-PARP, 1:1000 from Santa Cruz Biotechnology; rabbit anti-COX2, 1:1000; rabbit anti-iNOS, 1:1000 from Cell Signaling Technology) at 4°C overnight. On the second day, PVDF membranes were incubated with HRP-conjugated secondary antibody (anti-mouse and anti-rabbit, 1:2000) for 2 h at 37°C. Protein bands were detected by an ECL western blotting kit using a ChemiDoc-It™ imaging system (UVP, Upland, CA, USA). GAPDH was selected as a control. The gray value was analyzed by Gel-pro 32 (Media Cybernetics, Rockville, MD, USA).