Il 1β mouse mab

We analyzed specimens of normal human endometrial tissue using immunofluorescence. These experiments were approved by the institutional review board of Kyushu University (approval no. 622-00). First, we prepared 4 μm paraffin sections, allowed them to air dry, and then deparaffinized them. We retrieved the antigens via microwave heating in 0.1% sodium azide at pH 9.0. We allowed them to cool for 20 min at room temperature and then blocked them for 10 min using Protein Block Serum-Free (#X0909, Dako North America). The samples were then allowed to react overnight at 4 °C with the following primary antibodies: CD68 monoclonal antibody (1:100, #76,437, Cell Signaling Technology), IL1β Mouse mAb (1:100, #12242S, Cell Signaling Technology), and anti-IL17B (1:100, #NBP2-11672, Novus Biologicals, Englewood, CO, USA). After washing them with PBS, we incubated them for 30 min at room temperature with the following secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG(H + L) and Alexa Fluor 647 goat anti-mouse IgG(H + L) (1:1000 each, #A21206 and #A21236, Invitrogen/Thermo Fisher Scientific). We washed the reacted tissue with PBS, treated it with ProLong Gold antifade reagent with DAPI (#P36935, Invitrogen/Thermo Fisher Scientific), covered it with a glass coverslip, and imaged the fluorescence using a C2 confocal microscope (Nikon).