Dmi 6000 b wide field fluorescent microscope

To perform viability and mitochondrial staining, we transferred several tissue fragments per mouse into 96 well plates, and then incubated for 1 hour at 37 °C in a solution of DMEM with Calcein AM (Life Technologies) to label cytoplasm in live cells, NucBlue Live Cell Stain ReadyProbes reagent (Life technologies, Hoechst 33342 Special Formulation) to label nuclei, and MitoTracker Deep Red FM (Life Technologies) to label active mitochondria. Fragments were then rinsed in DPBS, transferred to a glass slide, sandwiched under a coverslip, and then imaged immediately using a Leica DMi6000b widefield fluorescent microscope. For dual live/dead staining, dead nuclei were stained using Ethidium homodimer and all nuclei were stained using Hoescht blue. Cells were imaged using a Leica DMi6000b widefield fluorescent microscope. Manual counting was performed on maximum intensity projection images of z-stacks (10X magnification), using the Leica LAS AF software.

Non-synchronized HeLa cells stably expressing H2B–eGFP and α-tubulin–mRFP were imaged every 4 min for 18 h with a 40× objective on a Zeiss Cell Observer HS inverted microscope equipped with a a Zeiss AxioCam MrX camera and temperature, humidity and CO₂ control systems. Images were processed and analyzed with FIJI software.
Immunofluorescence images were obtained with an inverted DMI-6000-B Leica wide-field fluorescent microscope equipped with 40× and 63× objectives and a Leica DFC 360FX camera. Confocal images were obtained on a Leica TCS SPE microscope equipped with a 63× objective and the following laser lines: 405 nm (for DAPI and Hoechst), 488 nm (for GFP and FITC), 532 nm (for mRFP, DsRED, Cy3, TRITC and TexasRed), 635 nm (for Cy5 and DRAQ5).

Images taken with a 63× objective on an inverted DMI-6000-B Leica wide-field fluorescent microscope, equipped with a Leica DFC 360FX camera, were analyzed using the FIJI program as follows.