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    1. The immunohistochemical specificity of anti-IL-1β (catalogue no. 12242; Cell Signaling Technology) has been validated by the manufacturer. Thus, by immunohistochemistry, it successfully detects IL-1β immunoreactive cells in human colonic sections from individuals with chronic colitis. Further, by western blotting, the same antibody detects mature recombinant human and mouse IL-1β and the human precursor (molecular mass of 34 kDa) in extracts of Raw 264.7 and human monocyte-derived THP-1 cells, following exposure to Brefeldin and lipopolysaccharide (LPS), respectively. Anti-IL-1β was replaced with diluent to act as negative controls. As further controls, paraffin sections of THP-1 cells, harvested after incubation with LPS (100 ng/ml for 3 h) and without (from Cell Signaling Technology), were evaluated in this laboratory for IL-1β immunohistochemical specificity. The immunostaining protocol for IL-1β was tested with formalin-fixed tonsil sections, supplied by Department of Surgery, University of Auckland. Sections from selected pancreas and tonsil were immunostained for IL-1β in two separate sections. They were then exposed to rabbit anti-CD68 or rabbit anti-CD3, followed by donkey anti-rabbit IgG-biotin and then streptavidin–Alexa 568. Pancreatic sections were immunostained subsequently for insulin as above, followed by donkey anti-guinea pig IgG–Alexa 488.

      IHC for IL-1b

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