Hoechst 33342 solution

HaCaT cells were incubated with 5 µg/mL sFLG for 30 d. After treatment, cells were plated in 24-well plates and stained with Hoechst 33,342 Solution (#861,405; Sigma-Aldrich). Bright field and Hoechst images were acquired in a Cytation 5 Reader (Biotek) using a 20 × objective and analyzed with ImageJ 1.53 (> 50 cells/treatment).

Different groups of adherent cells were washed with phosphate-buffer edsaline (PBS), fixed with 4% paraformaldehyde in PBS for 20 min, and permeabilized with 0.1% TritonX-100 for 5 min at room temperature. After further washing, the cells were incubated with rhodamine-phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min and a Hoechst 33342 solution (Sigma–Aldrich, St. Louis, MO, USA) was used to counter stain the nucleus for 10 min at room temperature in a dark room. Other cells were incubated with a mouse polyclonal antibody directed against Nrf2 or FN over night at 4 °C after blocking with 10% goat serum. Then the cells were incubated in the dark at room temperature for 1h with a secondary antibody (Alexa Fluor 488/Alexa Fluor555, Invitrogen, Carlsbad, CA, USA). The nucleus was stained with Hoechst33342 as aforementioned. Cells were placed under alaser scanning confocal microscope LSM710, CarlZeiss, Germany) for observation and image acquisition.

72 h after seeding, extracellular Flag-tags were immunostained in non-permeabilized cells. After culture medium removal, cells were washed once in ice cold PBS and incubated 1 h at 4°C with monoclonal anti-Flag antibody (2 μg/ml, Sigma-Aldrich # F1804). Then, cells were washed 3 times with ice cold PBS, incubated 20 min with 3% (w/v) paraformaldehyde (PFA) at 4°C and transferred to room temperature for the remaining staining procedure. Cells were then washed three times with PBS and incubated 1 h with an anti-mouse Alexa Fluor® 647 conjugated secondary antibody (2 μg/ml Molecular Probes #A31571). Cells were then washed 3 times with PBS, incubated with a Hoechst 33342 solution (200 ng/ml, Sigma #B2261) 1 h. Finally, cells were washed three times with PBS, immersed in PBS and incubated overnight before imaging.
All solutions were prepared in Dulbecco's PBS freshly supplemented with 0.7 mM CaCl₂ and 1.1 mM MgCl₂. Antibody solutions additionally contained 1% (w/v) bovine serum albumin (BSA, Sigma-Aldrich #A9056). All liquid handling was performed with a manual 96 channel pipette liquidator (Liquidator™ 96, Mettler Toledo #17010335). Solution volumes were 15 μl/well for antibodies, 25 μl/well for PFA and 50 μl/well for Hoechst.