Fluoview ver 3.1 viewer

Phosphoryl-NF-κB p65 localization was quantified via an immunofluorescence technique. Paraformaldehyde (v/v, 1/25) was used for IPEC-J2 cells fixation for 30 min at a temperature of 37 °C. After a PBS wash (0.1 mM, pH7.4), permeabilised in 0.5% Triton (Triton×100, Sigma, Harz Lower Saxony, Germany) for 20 min, and for 20 min blocked with 5% BSA, as well as hatched with the anti-phosphoryl-NF-κB p65 antibody (diluted 1:100) for the whole night at a temperature of 4 °C. After washing with PBS (0.1 mM, pH7.4) for the second time, the secondary antibody was used to incubate the cells for 1 h at room temperature. Coverslips were washed two times via PBS (0.1 mM, pH7.4), and hatched with the goat anti-rabbit IgG antibody for 1 h in the absence of light, and hatched in a DAPI staining solution for 10 min. After that it was washed again in PBS. The fluorescence was monitored using an Olympus-fluoview ver.3.1 viewer (Olympus Corporation, Miyazaki Prefecture, Kyushu, Japan) [38 (link)].

A confocal scanning fluorescence microscope (Olympus FV300, Tokyo, Japan) with a x 10 lens was used to visualize the distribution of live and dead bacteria throughout the biofilm. The live bacteria were observed after SYTO9 staining (LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Eugene, OR)) and bacteria with a compromised membrane were seen after staining in the dark at room temperature for 25–30 min with a propidium iodide (PI) solution (1.0 mg PI/mL (Sigma)). Scans through the biofilm were made at 5 µm intervals. An Olympus fluoview ver.3.1 viewer (Olympus, Tokyo, Japan) was used for analysis and image processing.