Fluoview f1000 confocal microscope

Immunocytochemistry was performed to verify the expression of basigin-2 in the RAW 264.7 cells. The cells were plated on Lab-Tek chamber slides (Nunc; Thermo Fisher Scientific, Rochester, NY) and incubated overnight at 37 °C with 5% CO₂. The cells were fixed with incubation in 4% paraformaldehyde in PBS for 15 min at room temperature and washed with several changes of PBS. The cells were incubated in 500 μl blocking solution (TBS containing 0.1% Tween-20; Sigma Chemical Company, St. Louis, MO; and 2% normal goat serum; Pierce/Thermo Scientific) overnight at 4 °C. The cells were incubated with an antibody specific for mouse basigin gene products ([10 (link)]; diluted to 1 μg/ml in blocking solution) for 1 h at 37 °C and then at 4 °C overnight. After washing with TBS, the cells were incubated with 250 μl of Alexa 488-conjugated goat-anti-rabbit secondary antibody (diluted 1:1,000 in blocking solution; Invitrogen Corporation) for 1 h at 37 °C. The cells were washed with TBS, with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen Corporation) added to the first wash. Coverslips were mounted with 30% glycerol containing p-phenylenediamine (Sigma Chemical Company), and the cells were viewed with an Olympus FluoView F1000 confocal microscope (Pittsburgh, PA).