Rabbit anti cox 2

Protein levels of TNF-α and COX-2 in the dorsal skin (n = 10 at each point in time) were analyzed according to our published procedure [17 (link)]. After sacrificing the animals, the tissues were homogenized in 50 mM PBS (pH 7.4) containing 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (pH 8.0), 0.2% nonidet P-40, 10 mM ethylenediaminetetraacetic acid (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium or thovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol (DTT). The skin tissues were separated using 4‒20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, and the resolved proteins were transferred to a nitrocellulose membrane for 2 h at 40 V. Each membrane was incubated separately with the primary antibodies: rabbit anti-TNF-α (1:1000, Abcam) and rabbit anti-COX-2 (1:1000, Abcam) overnight at 4 °C. The membranes were washed with a washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.05% Tween-20) and exposed to peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:1000 dilution at room temperature for 2 h. The membrane blots were developed using an enhanced luminol-based chemiluminescent (ECL) kit (Pierce Chemical, TX, USA).

After blocked with BSA for 30 minutes, sections (5 μm) of abdominal arteries were incubated with rabbit anti‐COX‐2 (1:100, Abcam), mouse anti‐CD68 (1:100, Abcam) or mouse anti‐α‐SM actin (1:100, Sigma‐Aldrich) overnight at 4°C. After incubated with secondary antibodies, a drop of Prolong Gold anti‐fade reagent with DAPI (Vector Laboratories) was used to seal the coverslip. Images were acquired by laser scanning confocal microscopy (LSM 710, Zeiss).

The following primary antibodies were used throughout this study: rat anti-mouse CD11b (1:400, Abcam), rabbit anti-F-actin (1:1000, Abcam), rabbit anti-COX-2 (1:1000, Abcam), rabbit anti-IL-1β (1:200, Abcam), rabbit anti-GFAP (1:5000, Neuromics), rabbit anti-Iba-1 (1:1000, Wako), goat anti-Iba-1 (1:500, Wako), rabbit anti-AKT (1:1000, Santa Cruz), rabbit anti-p-AKT (Ser473, Thr308) (1:1000, Cell Signaling), rabbit anti-ERK (1:1000, Santa Cruz), rabbit anti-p-ERK (Thr42/44) (1:1000, Cell Signaling), rabbit anti-STAT3 (1:1000, Cell Signaling), rabbit anti-p-STAT3 (Ser727, Abcam), mouse anti-PCNA (1:1000, Santa Cruz), rabbit anti-D2R (1:1000, Abcam), and rabbit anti-D1R (1:1000, Millipore) antibodies. We used the following small molecules: D1R antagonists (LE300, 10 μM, Sigma-Aldrich; SCH23390, 30 μM, Tocris), D1R agonist (A77636 hydrochloride, 10 nM, Tocris), D2R antagonist (eticlopride hydrochloride, 100 nM, Sigma-Aldrich), a STAT3 inhibitor (S3I-201, 50 μM, Sigma-Aldrich), and an ERK inhibitor (PD98059, 10 μM, Millipore).

After the NP cells were confluence in the six-well plate then lysed the cells with RIPA buffer (Millipore, Billerica, MA, USA) on ice for 40 minutes. Then centrifuged at 4°C, 12,000 rpm. Carefully absorb the supernatant and measure the protein concentration with BCA protein Assay Kit (Biotechnology Co., Beijing, China) according to the manufacture instructions. Separated each group of protein in 10% acrylamide–SDS–PAGE and subsequently transferred to PVDF membranes (Millipore, USA). Blocked the membranes with TBST which contain 5% milk powder and followed incubate with primary antibodies (rabbit anti-MMP-13, 1: 1,000, Abcam, USA; rabbit anti-COX-2, 1:1,000, Abcam, USA and rabbit anti-iNOS, 1:1000, Ancam) overnight at 4°C. Then incubate the PVDF membranes in goat anti-rabbit immunoglobulin (IgG)-horseradish peroxidase (HRP) secondary antibody (1:3000; Beijing Golden Bridge Bioechnology, China) at room temperature for 1 hour. Equal protein loading was confirmed by reprobing the membranes with the rabbit anti-GAPDH-HRP antibody (1:3,000, Proteintech, USA). Protein bands were detected using a FluorChem E Chemiluminescent Western Blot Imaging System (Amersham Imager 600, GE Amersham USA) and image-Pro Plus software (Media Cybernetics, Warrendale, USA) was used to analyze the optical density of each strip [23 (link)].

The intestine was isolated and fixed with 4% formaldehyde solution, then embedded in OCT. The cryosections were prepared. Then, the sections were permeabilized for 15 min with 0.1% Triton X-100 on ice and blocked in 5% BSA for 1 h at room temperature. The sections were then incubated overnight with the primary antibody at 4 ℃. Finally, the fluorescein-labelled secondary antibodies were applied for 1 h at room temperature. The following antibodies were used: rabbit anti-COX1 (Abcam, 1:200); rabbit anti-COX2 (Abcam, 1:200); mouse anti-PTGES (Santa Cruz, 1:50); rabbit anti-Muc2 (Abcam, 1:300); rabbit anti-Lyz (Abcam, 1:300); rabbit anti-Chga (Abcam, 1:300); rabbit anti-Olfm4 (CST, 1:300).