Ab9622

Specimens of submandibular glands for both IgG4-RD and controls and specimens of lymph nodes for controls were examined by immunohistochemical analysis of IgG, IgG4, IL-4, IL-13, IL-18 and IFN- expression. Three-µm-thick sections were prepared from each block of tissue embedded in paraffin. Deparaffinised sections were treated with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity. The sections were incubated with protein blocker (Dako, Glostrup, Denmark) for 30 min and incubated at 4°C overnight with rabbit anti-human IgG antibody(1:5000, Dako), mouse anti-human IgG4 antibody (1:2000, Life technologies, Grand Island, NY), rabbit anti-human IL-18 antibody (PM014, 1:2000, Medical and Biological Laboratories CO., LTD., Nagoya, Japan), rabbit anti-human IL-13 antibody (06-1090, 1:200, MILLIPORE, MA, USA), rabbit anti-human IL-4 antibody (ab9622, 1:200, Abcam, Cambridge, UK) and rabbit anti human IFN- antibody (ab9657, 1:500, Abcam) as primary antibodies. The sections were washed three times with phosphate-buffered saline (PBS, pH 7.2). After washing with PBS, the sections were exposed to Envision + secondary antibody (Dako) for 30 min. The reaction products were developed by immersing the sections in a 3′3-diamidobenzidine tetrahydrochloride solution. The sections were counterstained with hematoxylin.