Merck millicell ez slides

Cells (5 × 10³) were cultured in 6-well Merck Millicell EZ slides (Merck Millipore, Darmstadt, Germany), allowed to attach overnight, and treated with DOX for 24 h. Afterwards, the slides were washed twice in PBS, fixed in 4% formaldehyde in PBS (10 min, room temperature) and incubated with 0.2% Triton X-100 in PBS (10 min, room temperature). The fixed cells were incubated overnight at 4 °C with specific antibodies. Protein expression was detected using mouse monoclonal Ab against P-gp. The primary antibodies were detected after 1 h of incubation with anti-rabbit HRP-conjugated antibodies at a dilution 1:2000 in antibody diluent. Finally, the slides were washed 3 times in PBS and Pro Long Gold Mounting Medium with DNA intercalating dye 4,6-diamidino-2-phenylindole (DAPI) was added to visualize the cell nucleus. The analysis was conducted under fluorescence microscope.

Cells were cultured on six-well Merck Millicell EZ slides (Merck Millipore, Darmstadt, Germany) and allowed to attach overnight. After treatment, the slides were washed, fixed in 4% formaldehyde and permeabilized with 0.5% Triton X-100 in PBS, and stained with primary antibodies against IFITM1, STAT3 for 1 h. After washing three times, cells were stained with secondary antibody-conjugated fluorochromes for 1 h, and the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images were captured using the Leica SP5 II confocal laser-scanning microscope with Plan-Apochromat 63×/1.4 oil objective and LAS/AF Lite software for analysis.