Sc 1747

Western blot analysis was performed by lysing the cells in radioimmunoprecipitation assay buffer composed of 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) and containing a protease inhibitor mixture. The protein concentration was determined using the Bradford assay; the absorbance at 595 nm was measured on a microplate reader. An equal amount of protein for each sample was resolved by 8–10% SDS polyacrylamide gel electrophoresis and then electrophoretically transferred to a polyvinylidene difluoride membrane (Roche, Mannheim, Germany) that was blocked with 5% skim milk and incubated overnight at 4°C with primary antibodies against COX-2 (SC-1747; Santa Cruz Biotechnology, Santa Cruz, CA, USA), iNOS (#39898, Cell Signaling Technology, Danvers, MA, USA), NF-κB (#6956, Cell Signaling Technology), IκBα (#9242, Cell Signaling Technology), Akt (#9272, Cell Signaling Technology), p-Akt (#9271, Cell Signaling Technology), β-actin (#4967, Cell Signaling Technology, Danvers), and PCNA (SC-7907, Santa Cruz Biotechnology) used at 1:1000 dilution. The membrane was treated with horseradish peroxidase-conjugated secondary antibody for 1 h at 4°C, and proteins were detected by enhanced chemiluminescence (Animal Genetics, Tallahassee, FL, USA).

Fetal bovine serum (FBS) and modified Eagle’s medium (MEM) were purchased from Gibco (Grand Island, N.Y, USA), and phosphate-buffered saline (PBS), Western Breeze Chromogenic Kit anti-mouse, anti-rabbit, and anti-goat, and LysoTracker Green DND-26 were bought from Invitrogen (Carlsbad, CA, USA). Kit lactic dehydrogenase-based, sulfanilamide, H₃PO₄, and N-1-naphthylethylenediamine dihydrochloride were obtained from Sigma-Aldrich, St. Louis, MO, USA. Antibodies for IL-1β (H153): sc-7884, IL-2 (C2-1-hIL-2): sc-32295, IL-6 (E-4): sc-28343, IL-8 (807): sc-52870, IL-18 (H-173): sc-7954, tumor necrosis factor-alpha (TNF-α) (N-19): sc-1350, cyclooxygenase-2 (COX-2) (M-19): sc-1747, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) (H-300): sc-13032, matrix metalloproteinase-2 (MMP-2) (H-76): sc-10736, matrix metalloproteinase-9 (MMP-9) (M-17): sc-6841, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 (A): sc-109 and β-actin (C4): sc-47778 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Invitrogen (Carlsbad, CA, USA). All of the other chemicals used were of analytical grade and were purchased from Sigma (St. Louis, MO, USA).

Mammary tumors were frozen in OCT or fixed for 2 hours in 4% paraformaldehyde, sectioned and stained with hematoxylin and eosin as described previously [28 (link)]. For frozen sections, 5μm thick sections were fixed in acetone for 5′ at room temperature and stained with the following antibodies: K8 (1:200, Developmental Studies Hybridoma Bank, TROMA-1), CD45 (1:100, BD Biosciences, 550539), ADAM17 (1:200, Abcam, ab2051), F4/80 (1:100, BioRad, MCA49RT) and Cox-2 (1:200, Santa Cruz Biotechnology, sc-1747). For staining of paraffin-embedded tissues, the following antibodies and conditions were used. F4/80 (1:100; no antigen retrieval, MCA49RT, BioRad) and BrdU (1:300, sodium citrate antigen retrieval, Abcam, ab6326). For quantification of staining, total numbers of positive cells were counted in at least five 40x images taken from a minimum of three tumors per genotype.

Immunolocalization studies were performed using an indirect immunoperoxidase technique. Briefly, the renal tissue samples, 3–4 mm thick, were fixed by immersion in Bouin’s solution for 18–24 h at 21–24°C. Then the samples were dehydrated, embedded in Paraplast Plus, sectioned at 5–7 μm thickness with a rotatory microtome (Leica Biosystems, Germany), and mounted on glass slides. Kidney sections were dewaxed, hydrated and washed in Tris–phosphate buffer, pH 7.6 and incubated overnight with anti-COX-2 at 1:500 (SC-1747, Santa Cruz, CA), at room temperature. This was followed by incubation with the corresponding secondary antibody and with the peroxidase-antiperoxidase complex (MP Biomedicals, Santa Ana, CA). Peroxidase activity was detected by incubation of the sections with 0.1% (wt/vol) 3,3′-diaminobenzidine and 0.03% (vol/vol) hydrogen peroxide. Kidney sections were analyzed as previously described (Villanueva et al., 2008 (link)) using Nikon Eclipse E600 microscopy and Nikon DS-Ri1 camera (Nikon Corp., Tokyo, Japan). Four representative fields for each section were used and an average of six rats quantified. Images were quantified by Simple PCI 6.0 software (Hamamatsu Corporation, Sewickley, PA) and average values of stained areas were expressed as fold change of controls.

Immunofluorescence staining was performed to evaluate the nuclear accumulation of β‐catenin. The cells were grown to 40–60% confluency on sterile glass coverslips, fixed with 4% paraformaldehyde, washed once with phosphate buffered saline PBS, and soaked with 3% bovine serum albumin (BSA) for 30 min for blocking. Then, slides were incubated for 1 h at 37°C with anti‐β‐catenin rabbit polyclonal antibody (ab47426; Abcam, Cambridge, CA; 1:200 dilution) or anti‐COX‐2 goat polyclonal antibody (sc‐1747; Santa Cruz Biotechnology, Santa Cruz, CA; 1:500 dilution). After rinsing with PBS, fluorescent goat polyclonal anti‐rabbit IgG–H&L (Alexa Flour^® 488) (ab150077; Abcam; 1:1000 dilution) or donkey polyclonal anti‐goat IgG–H&L (Alexa Flour^® 488) (ab150129; Abcam; 1:1000 dilution) were used as secondary antibodies. After nuclear staining with DAPI(D1306; Life Technologies, Carlsbad, California), slides were analyzed under a fluorescence microscope. Nonimmune rabbit serum was substituted for the primary antibody as a negative control.