Bx60 wide field fluorescence microscope

Cells at 24 h after gas exposure were fixed in 4% paraformaldehyde for 10 min and blocked with 10% normal donkey serum (Sigma-Aldrich) for 1 h, followed by overnight incubation at 4 °C in primary antibodies: rabbit polyclonal anti-Ki-67 antibody (1:500; Abcam PLC, Cambridge, UK) and rabbit polyclonal anti-HIF-1α antibody (Novus Biologicals, Oxford, UK). Cells were incubated in Alexa flour 568-conjugated secondary antibody (ThermoFisher scientific) on the following day. Then, cells were costained for the cell nuclei with Vectashield mounting medium containing nuclear dye 4′,6-diamidino-2-phenylindole–mounting medium (Millipore). Slides were then examined using a BX60 wide-field fluorescence microscope (Olympus, Hamburg, Germany) and AxioCam camera (Zeiss, Oberkochen, Germany) with Zeiss software under 20× magnification. Images were captured using a camera. Fluorescence intensity was quantified as the mean pixel intensity of relevant antibody staining using Image J (version 1.52a, National Institute of Health). Ten representative regions per slide were randomly selected. Intensity values were calculated and expressed as relative to the control.

Cells were fixed in 4% paraformaldehyde for 10 min and blocked with 10% normal donkey serum (Sigma-Aldrich) for 1 h, followed by overnight incubation at 4 °C with one of primary antibodies; rabbit polyclonal anti-Ki-67 antibody (1:500; Abcam plc, Cambridge, UK), rabbit polyclonal anti-HIF-1α antibody (1:300; Novus biologicals, Abingdon, UK), rabbit monoclonal anti-MMP9 antibody (1:300; Cell signaling Technology, London, UK). Cells were subsequently incubated in Alexa flour 568-conjugated secondary antibody (1:300; ThermoFisher scientific, Waltham, MA, USA) and co-stained with Vectashield mounting medium containing nuclear dye 4′, 6-diamidino-2-phenylindole–mounting medium (Millipore, Burlington, MA, USA). Cells were visualized with a BX60 wide-field fluorescence microscope (Olympus, Hamburg, Germany) under a 20× magnification. Images were captured using a cooled AxioCam camera (Zeiss, Oberkochen, Germany) with Zeiss software. Fluorescence intensity was quantified as the mean pixel intensity of relevant antibody staining using Image J version 1.52a software (National Institute of Health).