Fluorescein isothiocyanate conjugated anti rabbit antibody

Caco-2 cells were plated onto glass slide chambers coated with poly-d-lysine (3x10 4 cells/well) and incubated for 24 h in the presence of rifaximin with or without ketoconazole.
After the treatment, cells were washed with PBS-Triton 0.1% (T-PBS), fixed in 4% paraformaldehyde and then incubated in 10% BSA/0.1% T-PBS solution for 90 min and for 1 h with a 10% BSA/0.1% T-PBS solution of anti-proliferating cell nuclear antigen (PCNA) antibody 1:100 (Abcam) for immunostaining. Finally, the cells were incubated for 1 h in the dark with fluorescein isothiocyanate conjugated anti-rabbit antibody 1:100 (Abcam). Nuclei were stained using hoechst stain (1:5,000) (Sigma-Aldrich) and images were captured using a camera (Nikon digital sight dS-U1) connected to a microscope (Nikon eclipse 80i; Nikon Instruments Europe). The analysis of RGB intensity was performed using NIH software and quantification of PCNA + proliferating cells was expressed as % of PCNA + expressing cells per selected area (1 mm 2 ).
Statistical analysis. Results were expressed as mean ± SEM of n= 4 experiments in triplicate. Statistical analysis was performed using parametric one way analysis of variance (ANOVA) and multiple comparisons were performed using Bonferroni's post hoc test. P-values <0.05 were considered to indicate a statistically significant result.