Blocking buffer

Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were rinsed twice with PBS. Blocking buffer (DakoCytomation, Glostrup, Denmark) was added for 30 min, and then stained with primary antibodies and fluorescent second antibody. The following antibodies were used: VEGFR2 (Cell Signaling, 9698S, 1:800 dilution), ERK 1/2 (Cell Signaling, 4696S, 1:100 dilution). Anti-mouse IgG (Alexa Fluor #594 Conjugate) (Cell Signaling, 8890, 1:2000 dilution), Anti-rabbit IgG (Alexa Fluor #488 Conjugate) (Cell Signaling, 4412, 1:2000 dilution).

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).

Formalin-fixed paraffin-embedded tumor tissue specimens were prepared as described previously [18 (link)]. For immunohistochemistry (IHC), the tumors were sliced into 4-μm-thick sections, deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01 M PBS, pH 7.4. Heat-induced epitope retrieval was performed using citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA), followed by blocking with a blocking buffer (Dako). The tissue sections were stained with anti-CD4 and CD8 antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies and 3,3′-diaminobenzidine substrate chromogen solution (DAB, Dako). IHC images were obtained using an Aperio ScanScope AT slide scanner (Leica Biosystems Inc., Buffalo Grove, IL, USA) and analyzed using ImageScope software 12.4.3 (Leica Biosystems, Buffalo Grove, IL, USA).

Zebrafish were euthanized with tricaine overdose prior to overnight fixation in 4% paraformaldehyde dissolved in PBS containing 120 µM CaCl₂ and 4% sucrose, pH7.4. The skin was manually removed with forceps, without disturbing the internal organs and the zebrafish were permeabilized with 0.5% TritonX-100 for 3 hr at room temperature. Following blocking with 5% donkey serum (Jackson Immunoresearch) in blocking buffer (Dako), samples were incubated in primary antibodies overnight at 4°C, washed 4x with 0.025% TritonX-100 containing PBS, incubated in secondary antibodies overnight at 4°C, washed 4x, incubated in an increasing glycerol gradient of 25, 50, and 75%, and mounted in VectorShield mounting medium. The following antibodies and dilutions were used: guinea pig anti-Insulin polyclonal (1:100, Thermo), rabbit anti-Somatostatin (1:100, BioRad), chicken anti-GFP (1:200, Aves), mouse anti-acetylated Tubulin (1:200, Sigma), mouse monoclonal anti-HuC/D (1:100, Cell Signaling). Secondary antibodies (Jackson ImmunoResearch) used in this study include donkey anti-guinea pig AlexaFluor647 and 405, donkey anti-rabbit Cy3 and AlexaFluor488, donkey anti-mouse AlexaFluor488 and 647, donkey anti-chicken AlexaFluor488. Nuclei were stained with 25 µg/ml DAPI. Images were taken on Zeiss LSM700 or LSM800 laser scanning confocal microscopes equipped with a 25x/NA0.8 objective.

Following surgical resection, tissues were fixed in 4% paraformaldehyde (4°C overnight). Tissues were cryoprotected in 30% sucrose/PBS then mounted in optimum cutting temperature medium. Tissue sections of 10 µm were cut on a Cryostat (Leica CM1860), incubated with blocking buffer (Dako, UK) for 1 hour, before primary antibodies were applied overnight (4°C). Antibody details are provided in online supplemental methods. Tissues were washed (PBS; 3×5 min), and species-specific secondary antibodies were conjugated to Alexa Fluor fluorescent dyes (1:400, Thermo Fisher Scientific, UK) applied for 1 hour, before washing (PBS; 3×5 min), mounting (Vectashield hard set mounting media, Vector Laboratories, USA) and cover-slipping. Controls with no primary antibody were used in all experiments to check for non-specific secondary antibody binding. Leica DM4000 epi-fluorescence microscope was used to visualise immunoreactivity of sections. To ensure uniformity of acquired images, all sections were orientated and cut in the same manner. Images were captured using MetaMorph software (Molecular Devices, UK) and prepared for figures using Photoshop (Adobe) and PowerPoint (Microsoft).