U mniba

Almost confluent cultures of HEK cells grown on poly-l-lysine-coated, glass-bottomed dishes were incubated in culture medium with dextran-conjugates of Texas Red (TR, 0.1 mg/ml, an inert marker) and Oregon Green (OG, 0.1 mg/ml, a pH indicator) for 12 h at 37°C to allow uptake of the indicators by endocytosis. After a further incubation (4 h) without indicators, the cells were washed with HBS and fluorescence was recorded in HBS at 20°C using an Olympus IX81 microscope with a 60x/1.45 NA objective. Cells were illuminated with a mercury xenon lamp using alternating filter sets: U-MNIBA (Olympus, λ_ex 470–495 nm, λ_em 510–550 nm for OG) and LF561A (Semrock, λ_ex 550–570 nm, λ_em 580–630 nm for TR). Images were captured at 2-s intervals using an EMCCD camera (Andor iXon 897) and analyzed using Cell∧R software (Olympus, Milton Keynes, U.K.). Records were corrected for background fluorescence determined under identical conditions from cells without indicators. Fluorescence changes from defined regions of interest (ROI) are expressed as F/F₀, where F₀ and F denote the average fluorescence within the ROI at the start of the experiment (F₀) and at each time point (F).

Chromosome images were directly captured through a cooled CCD camera (PXL1400, Photometrics, Tucson, AZ, USA) mounted on a fluorescence microscope (BX60, Olympus, Tokyo). The digitized images were analyzed using Chromosome image analyzing system ver. 4 (CHIAS IV) [46 , 47 ], for which publicly available image processing software, ImageJ (http://rsb.info.nih.gov/ij/) was used. CHIAS IV is the latest version developed specifically for detailed analyses of pachytene chromosomes [26 (link)]. Detailed image analysis steps of pachytene chromosomes using CHIAS III were described previously [29 (link), 48 (link)]. The CHIAS IV program and an instruction manual is available at: http://www2.kobe-u.ac.jp/~ohmido/index03.htm. We defined the parameters that represent chromatin compaction in pachytene chromosomes as the division of the estimated DNA content (Mb) of the region by the physical length in μm. Three fluorescence filters of U-MNIBA, U-MNG, and U-MNU (Olympus) were used for the detection of the individual fluorescence from fluorescein isothiocyanate (FITC), Cy3, and DAPI, respectively. Images were captured, merged and pseudocolored using IPLab Spectrum^™ (Version 2.4).

For observation of fluorescence of GFP, transformed cells were collected by centrifugation at 200×g for 10 min, and examined by a fluorescence microscope BX-60 (Olympus, Tokyo, Japan), using the cube U-MNIBA (Olympus). When GFP fluorescence was faint, we performed immunofluorescence detection using anti-GFP antibody, essentially according to Nishida et al. (2004) (link). In the reaction with antibody, an anti-GFP monoclonal antibody was diluted to 1/200 with immunoreaction enhancer solution (Can Get Signal immunostain solution B, TOYOBO, Osaka, Japan) and reacted for 1 h. As the secondary antibody, anti-mouse monoclonal antibody labelled with Alexa fluor 488 (Invitrogen, Carlsbad, CA) was diluted to 1/200 with immunoreaction enhancer solution, and reacted for 30 min. The immunostained cells were observed by the fluorescence microscope with the cube U-MNIBA (Olympus) for Alexa fluor 488, the cube U-MWIG (Olympus) for observation of autofluorescence of chlorophyll, and the cube U-MWU (Olympus) for observation of DAPI-stained DNA. Nomarski differential interference image was also recorded. All microscopic images were captured by a digital camera (model DP-70, Olympus).