Glucocorticoid Receptor Nuclear Translocation

Fibroblasts were cultured on μ-dish 35 mm (ibidi GmbH, Planegg, Germany) for 24 h, then stimulated with DMSO (vehicle control), 5 μM G75, or 1 μM DEX for 24 h. Cells were fixed, permeabilized, and blocked using Immunofluorescence application solution kit (Cell Signaling Technology, Danvers, MA, USA) according to manufacturer’s instructions. Cells were treated with primary antibody GR (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C and with secondary antibody (Alex Fluor 594, Cell Signaling Technology, Danvers, MA, USA) for 1 h at 22–25 °C. Cells were further stained with DAPI solution (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. Fluorescence microscopy was performed using a Nikon i2 U microscope (Tokyo, Japan). All images were processed in Nikon NIS-elements software (Ver. 4.0, Nikon, Tokyo, Japan). GR nuclear translocation was quantified as previously described by using Image J (NIH, Bethesda, ME, USA) [49 (link)]. Briefly, the percentage of the total corrected fluorescence of GR nuclear section per the total corrected fluorescence of total GR cellular section was calculated.