Nis elements viewer software

The cells were seeded in glass-bottom dishes and incubated for 24 h at 37 °C, 5% CO₂ atmosphere to allow cell adherence and growth. For real-time imaging, cell nuclei were counterstained with Hoechst 33342 (5 μg mL⁻¹) firstly and washed with PBS. Subsequently, confocal images were captured by a confocal microscope (A1R-Storm, Nikon, Japan) under 60× oil objective lens using 405 nm (nuclei, Hoechst 33342), 488 nm (OFF-ON module, BDP) and 561 nm (always-ON module, Cy3.5) lasers. The DAPI (450/35), FITC (515/30 nm), TRITC (605/75 nm) emission filters were used for Hoechst 33342, BDP and Cy3.5 imaging, respectively. Image acquisition was performed as soon as the addition of the culture medium containing BiRN (100 μg mL⁻¹), followed by real-time imaging for 30 min with a time interval of 1 min between frames. The raw images were processed by NIS-Elements viewer software (Nikon) and the ratiometric images were generated by ImageJ software (NIH).

The intestinal tissues were fixed with 4% paraformaledehyde, embedded in paraffin and cut into slice. After routinely dewaxing and hydration, the slices were conducted antigen heat retrieval in citrate buffer (0.01 M, pH 6.0), and then blocked with 10% donkey serum (containing 0.3% Triton X-100) for 45 min at room temperature. Sections were incubated with the following primary antibodies overnight at 4 °C: mouse monoclonal anti-mast cell tryptase antibody (1:500, ab2378, Abcam) or rabbit monoclonal anti-PAR2 antibody (1:400, ab180953, Abcam). After washing with phosphate buffer saline (PBS) for 5 min * 3 times, sections were respectively stained with donkey anti-mouse or rabbit Alexa Fluor 488 ((IgG H&L) secondary antibodies (1:300, Invitrogen) for 60 min at room temperature, and then washed with PBS for 5 min * 3 times. Finally, DAPI (1 μg/ml, Beyotime Biotech, China) were used for nuclei staining. Images were viewed and captured using a confocal laser scanning microscope (Nikon, Japan) with excitation wavelength appropriate for Alexa Fluor (488 nm or 594 nm), and analyzed using NIS Elements Viewer Software (Nikon, Japan).

Control and transcriptionally repressed HeLa cells were imaged with phase-contrast microscopy (Axiovert 200M; Carl Zeiss; 20 × objective lens; A-Plan Ph1; 0.3 NA) equipped with a CCD camera (CoolSNAP HQ2; Photometrics) at 37°C in DMEM supplemented with 10% FBS. To determine mitotic timing, images were captured every 15 min for 72 hr using the Micro-Manager 1.3 software (www.micro-manager.org). In the washout experiment, images were captured every 20 min for 24 hr. HeLa cells stably expressing GFP-Aurora B (LAP-Aurora B) were imaged with an inverted microscope (TE2000U; Nikon; 20 × objective lens; LWD; 0.4 NA) equipped with an electron-multiplying charge-coupled device (CCD) camera (iXonEM+; Andor Technology) at 37°C in DMEM phenol red free medium (Invitrogen) supplemented with 10% FBS and 25 mM HEPES. Eleven 1 µm separated z-planes covering the entire volume of the mitotic spindle were collected every hour for 6 hr using the NIS-Elements Viewer software (Nikon). Cyclin B1-Venus were imaged every 15 min for 7 hr using an IN Cell Analyzer 2000 microscope (GE Healthcare) at 37°C in phenol red DMEM free medium supplemented with 10% FBS. The duration of all drug treatments in live-cell experiments are schematically illustrated in each respective figure. All images were analyzed with open source image analysis software ImageJ and cell profiler.

The filters were stained using an MGG kit (Merck Millipore, Molsheim, France) according to the manufacturer’s instructions. Afterwards, filters were air-dried at RT for 5 min and stored protected from light until subsequent analysis by light microscopy using a Leica™ microsystem light microscope (10×, 20× and oil-40× objectives) and the NIS-Elements Viewer software (Nikon Instruments Inc.,Melville, NY, USA). The complete screening of the metal-rimmed filter, which can be performed retrospectively, takes 10 to 15 min for an experienced eye.