Alexa fluor 594 goat anti mouse secondary antibody

Cancer cells (1 × 10⁴ cells) were seeded in the glass coverslips and incubated in 5% CO₂ at 37 °C overnight. After treatment with drugs, cells were washed with PBS and fixed by 4% paraformaldehyde for 20 min at room temperature. The fixed cells were permeabilized with 0.05% Triton-X 100 in PBS for 20 min, and then blocked with SuperBlock (Thermo Fisher Scientific) in 4 °C overnight. After blocking, cells were incubated with NF-кB primary antibody for overnight and then with Alexa Fluor 594-Goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) and 4′,6-diamidino-2-phenylindole (DAPI, 1 mg/mL) for 1 h. The stained cells were washed and sealed with coverslips and the immunofluorescence images were acquired by Cell^R Xcellence fluorescence microscope system (Olympus, Japan).

For cell staining, cells grown on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in TBS (50 mM Tris–HCl, pH 7.4, 150 mM NaCl) buffer. For specimen staining, formalin-fixed, paraffin-embedded sections of pancreatic cancer tissues were obtained. After blocking with 5% BSA (Yeasen, Shanghai) in PBS, antibodies against O-GlcNAc (Abcam, Inc., ab2739) or CA19-9 (Abcam, Inc., ab3982) at a dilution of 1/200 in 5% BSA were used to detect their expression at 4 °C overnight. After washing, the samples were stained with AlexaFluor^® 488 goat anti-rabbit secondary antibody (Jackson ImmunoResearch, Inc.) or AlexaFluor^® 594 goat anti-mouse secondary antibody (Jackson ImmunoResearch, Inc.) at a 1/200 dilution for 1 h at room temperature. DAPI (Southern Biotech, Inc.) was used as the nuclear counterstain. Fluorescent images were acquired using a Leica confocal laser scanning microscope (Leica, Inc.). Confocal imaging was performed in seven random fields. Mean fluorescence intensity (MFI) was calculated using Image-Pro plus software (version 6.0, Media Cybernetics, Inc.).