Spectral confocal laser microscope

Fibroblasts were treated with the 940 nm diode laser in CW mode under different conditions (power of 0.2 or 0.5 W and energy density of 3 or 4 J/cm²) for 72 h at 37 °C. Immunostaining was performed on cells fixed in ice-cold methanol-acetone (1:1) for 10 min and then washed with PBS. Cells were subsequently blocked with FBS (10% in PBS) for 30 min and incubated for 2 h with the assayed mAbs (fibronectin and α-actin) at 1:500 dilution (Table 1). Nuclear counterstaining with 4,6-diamidino-2-phenylindole was performed after the removal of excess antibodies. Immunostaining was visualized using a Leica Spectral confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany).

The dermal fibroblasts were cultured in chamber slides (Sigma Chemical) at a concentration of 2 × 10⁴ cells/well. After a period of treatment of 24 h with punicalagin or ellagic acid at doses of 10⁻⁶ or 10⁻⁷ M. Cells were fixed in a cold methanol-acetone mixture (1:1) for 10 min and then rinsed with PBS. Following this, the cells were blocked with 10% FBS in PBS for 30 min and incubated for 2 h with the fibronectin or α-actin monoclonal antibody (mAb) at a 1:500 dilution, as outlined in Table 1. Following the removal of excess mAbs by washing, the cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Images of the stained cells were then captured using a Leica spectral confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Fibroblasts were treated with the selected doses of the different phenolic compounds during 24 h. The cells were fixed with a 1:1 ratio of ice-cold methanol-acetone during 10 min and then washed with PBS. Afterward, the cultures were blocked with 10% FBS diluted in PBS and incubated with the mAbs fibronectin and α-actin at a dilution of 1:500 (Table 1) for 2 h. Excess antibodies were subsequently removed, and nuclear counterstaining was performed using 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI). The staining was visualized using a Leica Spectral confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany).