Immunofluorescence application solution kit

HDFs were plated on to the 12-well plates which were preloaded with grass coverslips, followed by exposure to UVA irradiation (10 J/cm²). After UVA irradiation, HDFs were treated with or without DEQA and incubated for an additional 24 h. For general immunodetection, cells were fixed and stained, using immunofluorescence application solution kit (Catalog No. #12727; Cell signaling Technology, Danvers, MA, USA), based on manufacturer’s instructions. Briefly, cells were fixed in 4% formaldehyde for 15 min, and then immunostained with polyclonal antibody to collagen I (Catalog No. ab34710; Abcam, Cambridge, UK) at 4 °C overnight. The coverslips were washed and incubated with secondary antibody conjugated with Alexa Flour 488 (Catalog No. A-11008, Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. Coverslip slides mounted in ProLong Gold Antifade reagent with DAPI (Catalog No. #8961; Cell Signaling Technology, Danvers, MA, USA) for nuclear staining. Cells were observed under a fluorescence microscope (Olympus Corp., Tokyo, Japan).

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.

Detection of perilipin-1 and PPARγ expression in adipo-induced hBM-MSCs was observed by immunofluorescence staining. Cells were grown and induced to differentiate on glass coverslips . At day 10 of differentiation, cells were fixed and stained with anti-perilipin-1 (ab3526; Abcam) and anti-PPARγ antibody (ab9256; Abcam) conjugated with Alexa Fluor 488 (A-11008; Invitrogen) and ProLong Gold Antifade Reagent with DAPI (#8961; Cell Signaling Technology) to highlight the nuclei. Fixation and staining of the cells were carried out using immunofluorescence application solution kit (#12727; Cell Signaling Technology), according to the manufacturer's instructions.