Plan apochromat 63x 1.40 oil dic objective lens

After treatment with WABI, MM1R cells were fixed in 4% (v/v) formaldehyde. Cells were stained with rhodamine–phalloidin (Molecular Probes, Thermo Fisher Scientific Inc., Waltham, MA, USA) to visualize the cellular morphology, Hoechst 33,258 (Sigma-Aldrich, St. Louis, MO, USA) to visualize the nucleus and streptavidin-Alexa Fluor555 (Molecular Probes, Thermo Scientific, MA, USA) to visualize biotinylated WA. To determine WABI colocalization with specific proteins (BTK), WABI-treated MM1R cells were immunostained with primary (anti-rabbit BTK, Cell Signaling Technology, mAb#8547) and corresponding labeled secondary antibodies (anti-rabbit Alexa Fluor-488, Molecular Probes, Thermo Scientific, MA, USA). Cells were then placed on glass slides and mounted with coverslips using Fluoromount^TM (Sigma-Aldrich, St. Louis, MO, US)Confocal imaging was carried out by a laser-scanning microscope equipped with a Plan-Apochromat 63X/1.40 Oil DIC objective lens and excitation wavelengths 405, 488, 561 and 640 nm (Zeiss LSM 800, Carl Zeiss, Germany). At least 20 different microscopic fields were analyzed for each sample using ZEN imaging software (Carl Zeiss). ZEN lite^TM (Carl Zeiss, Germany) was used to perform image reconstruction and presentation.