Anti rabbit immunoglobulin igg alexa fluor 488

Cells covered with PBS were exposed to 40 mJ/cm² UVB (emission peak at 302 nm) using a CL-1000 M UV crosslinker (UVP, Upland, CA, USA), and the intracellular oxidative stress of Hs68 cells was quantified using DCFDA fluorogenic dye (Sigma-Aldrich, Saint Louis, MO, USA). Fluorescence was detected using a microplate reader (Synergy HTX, BioTek Instruments, Winooski, VT, USA) at emission and excitation wavelengths of 520 and 488 nm, respectively. For immunofluorescence staining, HS68 cells were fixed on coverslips with 4% paraformaldehyde after exposure to 40 mJ/cm² UVB. The slips were blocked with 5% skim milk in Tris-buffered saline (pH 7.6) containing 0.3% Triton X-100 and incubated with the following primary antibodies: anti-NFκB antibody (Cell Signaling, Danvers, MA, USA) and anti-4, 6-diamidino-2-phenylindole (DAPI) antibody (Cell Signaling, Danvers, MA, USA) for 30 min at 20 ± 1 °C. Following washing with PBS, the slips were incubated with the secondary-antibody anti-rabbit immunoglobulin (IgG) (Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA) for 30 min at 20 ± 1 °C. The cover slips were counterstained with ProLong Gold anti-fade reagent and DAPI (Thermo Fisher Scientific, Waltham, MA, USA), and images were captured using a fluorescence microscope (Leica DMIL, Wetzlar, Germany).

Human skin fibroblasts (Hs68 cells) were grown on a cover slip and treated with 0.25–2.0 mg/mL FAE after UVB irradiation. After 24 h, the cells were fixed with 4% paraformaldehyde and washed with PBS. They were then blocked with MPBS (containing 5% non-fat milk solution and 0.3% Triton X-100) for 30 min. PBS was again used to wash the coverslip, and incubation was performed with a primary antibody for 30 min and then with a secondary antibody, anti-rabbit immunoglobulin (IgG) (Alexa Fluor 488, Invitrogen, Carlsbad, CA, USA). Coverslips were then washed with PBS to remove unbound secondary antibodies, and counterstained using ProLong Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole. A fluorescence signal was observed using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).