Anti cox 2 antibody

Ethyl gallate (purity > 99%), arachidonic acid (AA), galangin, lipopolysaccharide (LPS; from Escherichia coli, serotype 055:b5), and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from GIBCO-Thermo Fisher Scientific (Waltham, MA, USA). The anti-COX-1 antibody and anti-COX-2 antibody were obtained from Abcam (Cambridge, UK), and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). The enzymatic immunoassay (EIA) kit for measurement of prostaglandin E₂ (PGE₂) was obtained from Cayman Chemical (Ann Arbor, MI, USA).

Five sections (5 μm in thickness) from each group were cut and adhered to slides treated with 3-aminopropyltriethoxysilane (APES (Sigma, USA)). Briefly, sections were deparaffinized with xylene and rehydrated in graded ethanol (100 to 70%). To minimize endogenous peroxidase activity, the slides were treated with 10% (v/v) H₂O₂ in water for fifteen minutes. The sections were washed with 0.01 M PBS (pH 7.2) and then blocked with 1% BSA, 0.2% Tween 20 in PBS for 1 h at room temperature. The sections were incubated overnight at 4°C with anti-TNF-α antibody (ABCAM, CA, USA, 1 : 250), anti-IL-1β antibody (GenWay, San Diego, CA, USA, 1 : 250), anti-COX-2 antibody (ABCAM, CA, USA, 1 : 400), and anti-iNOS (ABCAM, CA, USA, 1 : 50). The antigen-antibody reaction was visualized with avidin-biotin peroxidase (Dako Universal LSAB + Kit, Peroxidase), using 3,3-diaminobenzidine as the chromogen. The slides were counterstained with hematoxylin. Positive staining resulted in a brown reaction product. Five pictures at the same magnification were quantitatively analyzed using the Gimp 2.6 software program (GNU Image Manipulation Program, UNIX platforms).

Immunohistochemical staining with the VECTASTAIN ABC kit (Vector Laboratories, San Mateo, USA) was performed according to the manufacturer’s protocol. After antigen retrieval with EDTA (pH 9.0), inactivation in 3% H₂O₂/MeOH, and blocking with protein-block (Dako Japan), the antigen was incubated with rabbit polyclonal anti-COX-2 antibody (abcam, Cambridge, UK) at a 1:2000 dilution with Can Get Signal Solution 1 (Toyobo, Osaka, Japan). This was followed by incubation with the biotinylated secondary antibody and the VECTASTAIN ABC reagent.

Twenty-six days after LPS induction, IL-1β, IL-6, TNF-α, cyclooxygenase-2 (COX-2), and NF-κB concentrations in the hippocampus were assayed according to a previously described procedure (Lee et al. 2014 (link)). Three rats from each group were deeply anesthetized via isoflurane inhalation (1.2%), and were sacrificed one day after behavioral testing. The IL-1β, IL-6, TNF-α, COX-2, and NF-κB concentrations were evaluated with competitive enzyme-linked immunoassays (ELISAs) using anti-IL-1β antibody (Abcam, Cambridge, MA, USA), anti-IL-6 antibody (Abcam), anti-TNF-α antibody (Abcam), anti-COX-2 antibody (Abcam), and anti-NF-κB antibody (Abcam) according to the manufacturer's protocols.

Tissues were fixed with 4% paraformaldehyde for 8 h and then dehydrated and embedded in paraffin. They were cut at a thickness of 5 µm. The sections were dewaxed and treated with 0.01 M citrate buffer at 80°C for 20 min for antigen retrieval, and then blocked with 10% horse serum for 1 h. Sections were then incubated for 24 h at 4°C with anti-CBP antibody (1∶200; Santa Cruz) or anti-p300 antibody (1∶200; Santa Cruz), and then incubated with biotinylated anti-mouse IgG (1∶200; Santa Cruz) for 2 h, followed by red dihydroxyfluorane (1∶100; Jackson) incubation for 2 h. Sections were then blocked with 3% goat serum for 1 h, incubated with anti-Cox-2 antibody (1∶200; Abcam) or anti-BDNF antibody (1∶200; Santa Cruz) overnight at 4°C, followed by biotinylated anti-rabbit IgG (1∶500; Santa Cruz) for 2 h and green dihydroxyfluorane (1∶200; Jackson) incubation for 2 h. Sections without primary antibody served as the negative controls. Sections were then scanned with a Leica confocal laser scanning microscope (TCS SP5, Mannheim, Germany).

COX-2 protein expression was evaluated in RAW 264.7 cells by immunocytochemistry. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and stained overnight at 4°C with rabbit polyclonal anti-COX-2 antibody (1 : 400; Abcam, Cambridge, UK). Subsequently, cells were washed with PBS three times and incubated with fluorescent isothiocyanate-conjugated donkey anti-rabbit IgG (1 : 500; Jackson ImmunoResearch). The slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Inc.) and examined using a confocal microscope (LSM5 PASCAL; Carl Zeiss) equipped with a filter set with excitation at 488 and 543 nm.