Leica dfc300 fx camera

Cells (2×10⁴ per slide) were cytospun onto glass slides for 15 min at 1,000 rpm (Cytospin; Thermo Shandon, USA). Cells were fixed at room temperature in methanol for 5 min and air-dried. Fixed cells were stained with May-Grunwald stain followed by Giemsa stain per the manufacturer's protocol (Sigma). Light microscopy images were obtained using a Leica DM LB2 microscope, Leica DFC 300FX camera, and Leica Application Suite 3.1.0 software (Leica Microsystems, Switzerland). Blinded erythroid differential counts were performed by a hematopathologist, at Sunnybrook Health Sciences Centre, University of Toronto. A total of 6 cytospin slides with May-Grunwald Giemsa stains were prepared during two separate experiments for each non-transduced, double-sorted MigR1, and MigR1-Fli-1 DP17-vector cell groups. Approximately 100–200 cells were counted on each slide and categorized into one of the three defined stages; R1, proerythroblast; R2, early basophilic erythroblast; R3, late basophilic erythroblast. Data are presented as the percentage of total cells analyzed.

HK-2 cells were washed twice with PBS for 5 min each. The cells were fixed with 4% paraformaldehyde for 15 min, blocked with goat serum for 1 h, and incubated overnight with primary antibodies against SIRT3 and cubilin (Biorbyt, Cambridge, UK). Then, the slides were incubated with goat anti-rabbit IgG/FITC (1:100; Proteintech, China). Nuclei were stained with DAPI (Solarbio). Fluorescence was captured using a Leica DMI4000 B automated inverted microscope equipped with a Leica DFC300 FX camera (Leica, Wetzlar, Germany) and evaluated using Image-Pro Plus 6.0 analysis software (Media Cybernetics, Rockville, MD, USA).

The fluorescently tagged embryos were live imaged on Nikon Widefield Epi-fluorescent inverted microscope (Nikon Ti inverted fluorescent Microscope with CSU-W1 large field of view) supported by ANDOR iXon camera or Nikon TIRF/Spinning Disk microscope (Nikon Ti inverted fluorescent Microscope with CSU-22 spinning disc confocal) supported by Prime 95B Scientific CMOS camera at 37C°. After live-imaging, these samples were subsequently performed ISH and imaged by Leica MZ16F microscope with Leica DFC300 Fx camera and Leica FireCam V.3.4.1 software. BrdU-stained samples were imaged on Nikon Ti2 inverted fluorescence microscope supported by a Crest LFOV spinning disk confocal.