Immunofluorescent Localization of 4-1BB and Gal-9

2 × 10⁵ cells were immobilized on poly-l-lysine–coated glass coverslips (BD) for 30 min, fixed with 4% paraformaldehyde (PFA)/PBS (GE Healthcare) for 15 min, and permeabilized with 0.3% saponin/PBS for 5 min. After blocking with 5% BSA/PBS, rat anti–mouse 4-1BB 3E1 clone (Shuford et al., 1997 (link)) and rabbit anti–mouse Gal-9 (Novus Biologicals) antibodies were treated at room temperature for 1 h, followed by anti–rat IgG–Alexa Fluor 555 (Cell Signaling Technology) and anti–rabbit IgG–Alexa Fluor 488 (Invitrogen), with three PBS washing steps in between. After treatment with DAPI for 10 min, for visualizing the nuclei, cells were mounted with FluorSave Reagent (EMD Millipore). Immunofluorescence was analyzed at ×60 with an Axiovert 200M microscope (Carl Zeiss) integrated with Intelligent Imaging Innovations SlideBook 4.2 software.