Anti il 1

The specimens were incubated with a blocking solution (1% BSA, 0.5% Tritonx-100, and 10% normal goat serum in PBS 0.1M), and then the slices were incubated overnight at 4 °C with a solution containing rabbit polyclonal anti-VEGF-C primary antibody (1:50, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-IL-1ß (1:50, Santa Cruz Biotechnology, Inc.) or anti-PDGFR (1:100, Abcam, Cambridge, UK) or anti-TGF- ß (1:100, Bioss Antibodies, Woburn, MA, USA) or pSTAT3 (1:100, Cell Signaling, Beverly, MA, USA). All the primary antibodies used cross-reacted with human tissue. All specimens were incubated at room temperature for 2 h in labeled conjugated goat anti-rabbit secondary antibodies (AlexaFluor488 or 546, IgG, Invitrogen, Waltham, MA, USA) diluted 1:400 in 0.1 MPBS and DAPI stained (1:500 in 0.1 M PBS). Images (20 or 60×) were obtained with a confocal laser scanning system (NIKON TE, Confocal Head A1 MP, Tokyo, Japan) with an Ar/ArKr laser (for 488 nm excitation) and a HeNe laser (for 543 nm excitation). DAPI staining was imaged by two-photon excitation (740 nm, o140 fs, 90 MHz) performed with an ultrafast tuneable mode-lockedTi: sapphire laser (Chameleon, Coherent Inc., Santa Clara, CA, USA).

Protein samples (30 µg of protein/sample) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then blotted onto nitrocellulose membranes (90 min at 110 V) using standard procedures. Membranes were blocked in PBST (PBS with 0.1% Tween) containing 5% non-fat dry milk for 90 min and incubated overnight at 4 °C with primary antibodies: anti-ß-galactosidase (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-65670, RRID:AB_831022IBA1), anti-IκBα (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-1643, RRID:AB_627772), and anti-IL-1ß (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-52012, RRID:AB_629741). The day after, blots were rinsed three times with PBST and incubated for 2 h at rt with HRP-conjugated secondary antibodies and then detected by chemiluminescence detection system (Life Technologies Italia, Monza, Italy). Signal intensity (pixels/mm²) was quantified using ImageJ (NIH). The signal intensity was normalized to that of GAPDH (1:5000 Santa Cruz Biotechnology, Dallas, TX, USA) [25 (link)]. The treatments were carried out in three independent experiments (n = 4), and protein expression was calculated by normalizing the values to the mean of the control [20 (link)].