Rabbit anti mouse cox 2

Immunohistochemistry (IHC) was performed manually or automatically with an autostainer (BenchMark XT, Ventana Medical Systems Inc., Tucson, AZ, USA). For the manual protocol, paraffin sections were first deparaffinized and rehydrated in ethanol/water solutions. Epitopes on tissue were then retrieved with Heat-Induced Epitope Retrieval (HIER) in citrate buffer (0.01 M, pH 6.0). For blocking endogenous peroxidase activity, sections were treated with 3% hydrogen peroxide for 30 min in the dark. To reduce non-specific primary antibody binding, Blocking Buffer (DAKO) was used for one hour at room temperature. Sections were then incubated with primary antibodies at 4 °C overnight. Rabbit anti-mouse COX-2, anti-human cPLA2, and anti-8-OHdG polyclonal antibodies were purchased from Santa Cruz Biotechnology (SC-1747-R, SC-7891, and SC-139586, Santa Cruz, CA, USA). Afterward, staining was detected with a DAKO polymer system. For image acquisitions, three random high-power magnification fields were obtained in each sample by a Nikon Digital Camera Microscope (Nikon, Tokyo, Japan). All slides were reviewed by a blinded pathologist (Dr. Shih-Hao Liu), and the area percentage of staining in a 200× power magnification field were analyzed by NIH ImageJ software (Version 1.47, Bethesda, MD, USA).

Cells were co-cultured using the transwell method. Protein was extracted from RAW264.7 cells cultured in the upper chamber and from 3T3-L1 cells cultured in the lower chamber. For western blot analysis, cells were lysed using M-PER Mammalian Protein Extraction reagent (Pierce, Rockford, IL) with a protease and phosphatase inhibitor cocktail (Nacalai Tesque). Protein concentration was determined using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Denatured proteins were separated using SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and then transferred onto Immobilon-P membranes (Millipore, Billerica, MA).
The following antibodies were used: rabbit anti-mouse HRASLA3 (AdPLA) (1:1000; BD Biosciences, Franklin Lakes, NJ), rabbit anti-mouse COX2 (1:1000; Santa Cruz Biotechnology), rabbit anti-mouse PRKAR1A (1:1000; GeneTex, Irvine, CA), rabbit anti-mouse actin (1:1000; Santa Cruz Biotechnology), and anti-rabbit horseradish peroxidase–conjugated immunoglobulin G (1:2000; GE Healthcare, Bucks, UK). The blots were developed using ECL (GE Healthcare). Relative protein expression levels were determined by dividing the band intensity of the product of interest by that of the actin control band. The intensity of the level of objective protein expression was analyzed using ImageJ software (http://imagej.nih.gov/ij/) [38 (link)].