Goat isotype control

Solid tumor samples were snap-frozen in OCT medium (Tissue Tek, Sakura, Torrance, CA) and sections were prepared using a Leica CM1950 Cryostat (Leica Microsystems, Bannockburn, IL). Sections were fixed in cold acetone for 10 minutes, pretreated with 3% H₂O₂ for 20 min to block endogenous peroxidase activity and blocked in normal horse serum (Vector Laboratories). Biotinylated goat anti-mouse Exodus-2/CCL21 and goat isotype control (both R&D systems) were used at 1:50 dilution for these studies. Then, the Vectastain ABC kit was applied as described by the manufacturer (Vector Laboratories). Sections were counterstained with Gill's hematoxylin (Vector Laboratories). Images were acquired through a Micropublisher 5.0 Digital CCD Color Camera (Qimaging, Surrey, BC Canada).

Confluent ARPE-19 cells were cultured for 3 weeks, serum starved (1% FBS) overnight, then treated with IL-1 β (5ng/ml) alone or IL-1 β plus NVS-ZP7-4 (10 μM) for 2hr or 24hr before harvesting for FACS analysis. Cells were harvested and diluted to 1X10⁶ cells per ml in FACS buffer (PBS; 0.1% BSA), total of 2X10⁵ cells per well were added to each well of a 96-well plate and centrifuged at 400Xg for 3 min at 4°C before removing the supernatant. 50 μl of human IL-1RI FITC-conjugated Antibody (R&D) or goat isotype control (R&D) was added to the cell pellets and incubated for 30min at 4°C. The cells were washed and pelleted twice with 200 μl FACS buffer then resuspended in 200 μl FACS buffer and fluorescence values were measured with a BD FACSCanto II cytometer (BD Biosciences, Franklin Lakes, NJ). The amount of cell surface-bound anti-IL1R1-FITC was assessed by measuring the mean channel fluorescence of 10K collected cell events. Cells were initially gated using forward and side scatter properties to isolate single cells. Data for each sample were then plotted on a histogram with FITC-positive cells having values above the negative isotype control sample (not shown). Data were analyzed using FlowJo7.5.5. Mean fluorescence intensity (MFI) or percentage of positive cell were plotted in the graph to compare level of expression of IL-1R1.