Slowfade diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi

GMSCs (3 × 10⁴ cells/well) were seeded onto glass coverslips and stimulated with the indicated amounts of TNF-α and IFN-α for 6 and 24 h to observe the nuclear accumulation of HIF-1α. The cells were washed twice with PBS (pH 7.4), fixed with 4% paraformaldehyde for 20 min, blocked with Blocking One Histo (Nacalai Tesque) for 5 min at room temperature, treated with 0.5% Triton X-100 (Junsei, Tokyo, Japan), which penetrated into the cells, for 10 min at room temperature, and stained with primary anti-HIF-1α (1:200, D1S7W, Cell Signaling Technology) antibody for 1 h at room temperature. Immunofluorescent labelling of HIF-1α was performed using anti-HIF-1α (1:200, D1S7W, Cell Signaling Technology). Subsequently, Alexa Fluor® 488 secondary antibody (1:1000; Life Technologies) was used as secondary antibody for 1 h in the dark at room temperature. Nuclei were stained using SlowFade® Diamond Antifade Mountant with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Waltham, MA, USA). Images were captured using a confocal laser-scanning microscope (Carl Zeiss LSM 700; Oberkochen, Germany) and ZEN 2012 software.

Cells were seeded onto sterile round coverslips coated with poly-L-lysine (Thermo Scientific, US), treated with 25 µM coralyne and exposed to IR according to the scheme of the experimental procedure (Figure 11). Thereafter, cells were fixed using an Image-iT^® Fixation/Permeabilization Kit (Life Technologies, US). Briefly, cells were fixed for 15 min with PBS (pH 7.3) containing 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100 for 15 min, and blocked for 1 h with Dulbecco’s PBS (pH 7.4) containing 3% bovine serum albumin (BSA). The organization of the F-actin network was evaluated in cells stained with rhodamine phalloidin (Invitrogen, US) diluted 1:300 with 0.1% BSA for 20 min at room temperature. The organization of the integrin-β1 network was analyzed in cells stained with an anti-integrin-β1 antibody conjugated with FITC (Abcam, Cambridge, UK) diluted 1:100 with 0.1% BSA for 60 min at room temperature. After staining, cells were washed three times with PBS for 10 min and mounted with SlowFade™ Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA, USA) to visualize nuclei. Fluorescent signals were observed with a confocal laser scanning microscope (Nikon A1) equipped with a Nikon Fluor ×60 water immersion objective.