Epr19025 42

MSLN protein expression was examined by immunohistochemical staining in MSLN cDNA-transduced iPSDCs and ubiquitin-MSLN combined cDNA-transduced iPSDCs. iPSDCs were collected using Smear Gell (GenoStaff, Tokyo, Japan). The endogenous peroxidase activity was suppressed by a solution of 0.3% hydrogen peroxide in methanol for 30 min. After being rinsed in Tris-buffered saline (TBS), the sections were incubated with a blocking reagent; Protein block (Dako, Kyoto, Japan) for 20 min at room temperature. The sections were incubated for 30 min at room temperature with the primary antibody, a 1:1500 dilution of an anti-human MSLN monoclonal antibody (EPR19025-42, Abcam, Cambridge, UK). After rinsing in TBS, the primary antibody was visualized using anti-rabbit antibody (Dako) for 60 min at room temperature, and DAB kit (Nichirei, Tokyo, Japan).

PE-conjugated anti-human CD11c (Becton Dickinson) was used for monocyte-derived DCs (MoDCs) and iPSDCs staining. Intracellular staining with an anti-human MSLN monoclonal antibody (EPR19025-42, Abcam) and Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody (Abcam) was performed using a Fixation and Permeabilization Solution Kit (Becton Dickinson).