Figure 4.

Immunohistochemical analysis of tobramycin and CuT uptake by HLMVEC and the effect of pretreatment with heparitinase II. (A) Confocal laser scanning microscopy (CLSM) images of untreated HLMVEC (1) and HLMVEC treated with increasing concentrations of tobramycin at 0.01 mM (2), 0.1 mM (3), 0.5 mM (4) before and after (5) heparitinase II treatment and copper-tobramycin at 0.01 mM (6), 0.1 mM (7), 0.5 mM (8) before and after (9) heparitinase II treatment. HLMVEC were pretreated with heparitinase II (5 U mL⁻¹) for 30 min, followed by treatment with tobramycin or copper–tobramycin (0.01–0.5 mM) for 3.5 h at 37°C and under 5% CO₂. Scale bar 100 μm. (B) Calibration bar of total fluorescence intensity. (C) Fluorescence intensity histogram of Figure 4, panel 4. (D) Fluorescence intensity histogram of Figure 4, panel 5. (E) The fluorescence intensity for each condition was analysed as the mean of at least three fields of view. The data expressed as mean ± SEM were analysed using one-way anova and Dunnett's post hoc test versus the negative control and *P < 0.05, **P < 0.01, ***P < 0.001. Uptake following heparitinase treatment was compared with uptake of 0.5 mM tobramycin and CuT by untreated cells using a Student's two-tailed unpaired t-test, with ^#P < 0.05, ^##P < 0.01. Representative images from a single experiment, typical of at least three independent experiments, are shown.