Anti IL-18

In vitro neutralization. Spleens were aseptically removed and cell suspensions were prepared. Cells were washed twice and resuspended in RPMI supplemented with 10% FBS. For cytokine measurement, spleen cells were cultured at 5 mln/mL in 24 well, flat bottom culture plates in the presence of several dilutions of rabbit anti murine IL 18 antibody (1:400; 1:200; 1:100; 1:50) and 100 ng/mL of LPS (a phenol extracted preparation from Escherichia coli 055:B5, Sigma Chemical Co). Cultures were incubated at 37C in a humidified atmosphere with 5% CO2. At the end of the incubation period, cultures were frozen at 70C and subjected to 3 freeze thaw cycles to obtain total cytokine levels. Before assaying, samples were centrifuged for 10 minutes at 10,000g to remove debris.

Immunofluorescence microscopy of IL-18 in mouse colon sections. The transversing portion of the large intestine from DSS-exposed (Panel A) and -unexposed mice (Panel B) was excised, rinsed in PBS, and frozen on isopentane cooled with liquid nitrogen. Frozen sections (5 μm) were cut on a Leica CM 1850 cryostat. The slides were fixed for 10 min in 4% paraformaldehyde, air-dried, and incubated for 20 min in PBS supplemented with 10% normal goat serum. Sections were incubated in a 1:50 dilution of Rockland's rabbit anti-Mouse IL-18 antibody or 1 μg/ml nonimmune rabbit IgG (not shown) as negative control. The antibodies were diluted in PBS containing 1% bovine serum albumin. After an overnight incubation at 4°C, the sections were washed three times with 0.5% bovine serum albumin in PBS. The sections were then incubated with a secondary goat anti-rabbit antibody conjugated to Alexa488 (Molecular Probes) for 60 min at room temperature in the dark. Nuclei were counterstained blue using 1 μg/100 ml bisbenzimide. After staining, sections were washed and examined with the Leica DM RXA confocal laser scanning system and analyzed. Similar staining will occur with other systems.

Immunohistochemistry with Rabbit anti-Mouse IL-18 antibody showing IL-18 staining in inflammatory cells of the mucous corium of mouse colon at 20x and 40x. Slide A is a negative control. Slides B and C show staining. Formalin fixed/paraffin embedded sections were subjected to heat induced epitope retrieval (HIER) at pH 6.2 and then incubated with mouse anti-IL-18 antibody at 4.0 ug/ml for 60 minutes. The reaction was developed using MACH 4 universal AP polymer detection system and visualized with WARP RED.