Caco-2 monolayers were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min. Then, the cells were blocked by incubation with PBS containing 3% BSA for 1 h at room temperature. Next, they were incubated with primary antibodies, including ZO-1 (1:50, BD), Occludin (1:50, R&D), Claudin-1 (1:50, Abcam) and NF-κB p65 (1:50, Santa cruz), at 4℃ overnight and then with secondary antibodies (diluted 1:400) for 1 h in the dark. Subsequently, the cells were washed and mounted using ProLongR Gold Antifade Reagent with DAPI (Invitrogen). Fluorescence was visualized by laser scanning fluorescence microscopy (Leica TCS SP5; Leica).
Prolongr gold antifade reagent with dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLongR Gold Antifade Reagent with DAPI is a mounting medium used in fluorescence microscopy. It is designed to preserve fluorescent signals and reduce photobleaching. The product contains DAPI, a fluorescent dye that binds to DNA, allowing for the visualization of cell nuclei.
Automatically generated - may contain errors
2 protocols using prolongr gold antifade reagent with dapi
1
Immunofluorescence Analysis of Tight Junctions
2
Immunocytochemistry Assay for Chibby, β-catenin, and Ki67
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Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 5 min, permeabilized with 0.2% Triton X-100 in PBS for 15 min, rinsed three times with PBS, and blocked with normal goat serum diluted 1:20 or with 5% BSA/PBS for 30 min. Staining was performed with primary rabbit polyclonal Ab to Chibby (AP9148b, Abgent, San Diego, CA, USA), mouse monoclonal Ab to β-catenin (A5441, Sigma, St. Louis, MO, USA), or rabbit polyclonal Ab to Ki67 (NB110-89717, Novus, Littleton, CO, USA) at a dilution of 1:100 at 4 °C overnight. Cells were washed five times with PBS and then incubated with a donkey anti-mouse Texas Red –conjugated secondary antibody for 2 h (Molecular Probe) at room temperature. Finally, the slides were mounted by ProLongR Gold antifade reagent with DAPI (Invitrogen, Eugene, OR, USA) and examined using a Zeiss Axiophot microscope (Zeiss Inc., Boston, MA, USA).
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