710 confocal microscopy

The quantification of intracellular ROS was based on the oxidation of dihydroethidium (DHE, Thermo Fisher). Briefly, normal and TOC keratinocytes (2 × 10⁵) were plated in each well of six-well plate. Cells were then washed twice with PBS and incubated with DHE (5 µM) at 37 °C for 30 min in the dark. After incubation, the cell were washed twice with PBS and were analysed by a BD FACSCanto ІІ Flow Cytometer (BD, UK). Data represented the value of ROS in the live cells by a ratio referred to the control. For staining, normal and TOC keratinocytes (5 × 10⁴) were plated on cover slip in each well of 12-well plate. The cells on cover slip were washed twice with HBSS after exposure to DHE (Thermo Scientific) working concentration (5 µM) at 37 °C for 30 min in the dark. Before microscopy, Hoechst 33342 (0.1 μg/ml, Thermo Scientific) was added to stain the nuclei. The fluorescence was analysed using Zeiss 710 confocal microscopy (Carl Zeiss).

PLA was performed using the Duolink in situ kit (Sigma) according to the manufacturer’s instructions. Cells were plated on coverslips in 12-well plates and after 24 h fixed with methanol acetone or PFA. Following fixation, cells were incubated in Duolink blocking solution for 30 min at 37 °C. Primary antibodies were diluted in Duolink Antibody diluent and added to the cells overnight at 4 °C. The following day the cells were washed in Wash buffer A two times for 5 min. The PLA plus and minus probes were diluted in antibody diluent and added to the cells, then incubated for 1 h at 37 °C. The cells were washed in Wash buffer A two times for 5 min. The ligation-ligase was prepared according to instructions and applied to cells for 30 min at 37 °C. After washes, amplification with Duolink Amplification-Polymerase solution was performed for 100 min at 37 °C. The cells were washed in Wash Buffer B two times for 10 min and then mounted with Duolink in situ mounting medium with DAPI before visualisation with Zeiss 710 confocal microscopy (Carl Zeiss). As a positive control binding between K6 and K16 was analysed, while as a negative control no primary antibodies were applied. Quantifications were performed using Image J Software.

Immunohistochemistry was performed on 5 mm frozen tissue or on cells plated in cover slips; sections were air-dried before processed. Cells/tissues were fixed in 4% paraformaldehyde (PFA) or in ice cold methanol–acetone (50:50 mixture) at room temperature for 15 min. If PFA fixation was used, samples were permeabilized with 0.1% Triton X-100. Cells/tissues were washed three times with PBS for 5 min each and incubated with 5% goat serum in PBS for 1 hour at room temperature to reduce nonspecific binding. After the cells/tissue were incubated with primary antibody in 5% goat serum overnight at 4 ˚C. The following day cells/tissues were washed three times with PBS and incubated with the secondary antibody conjugated with Alexa Fluor (Molecular Probes) in 5% goat serum for 1 h at room temperature. After three washes, cells/sections were incubated for 10 min with DAPI (100 ng/ml). Cells/tissues were mounted onto slides using Vectashield Mounting Medium (Vector Laboratories). Fluorescence was evaluated in one single plane by Zeiss 710 confocal microscopy (Carl Zeiss).