Nis elements documentation software

Samples were sectioned into 6‐μm‐thick cross‐sections using a microtome and stained with Toluidine Blue O. Sections were examined at × 40 and × 100 (oil) using a Nikon Eclipse 50i Compound Microscope (Nikon, Tokyo, Japan). A Nikon DS Camera Control Unit DS‐U2 and DS‐5M Camera Head were used to capture images, which were then processed using NIS Elements Documentation software (Nikon).

Fluorescent and bright-field images were recorded using an AE31 inverted Epi-fluorescence microscope (Motic, Causeway Bay, Hong Kong) with an IS1000 eyepiece (Tucsen, Fujian, China) or an Eclipse 80i Epi-fluorescence microscope (Nikon, Tokyo, Japan) with the NIS-elements documentation software (Nikon). For the AE31 fluorescent microscope, excitation filters were set at 480/30, 350/50, and 560/40 nm for GFP, blue (BFP), and red fluorescent protein (RFP) channels, respectively. Emission filters were set at 535/40, 460/50, and 630/60 nm for GFP, BFP, and RFP channels, respectively. For the Eclipse 80i Epi-fluorescent microscope, excitation filters were set at 450–490, 340–380, and 510–560 nm for GFP, BFP, and RFP channels, respectively. Emission filters were set at 520, 435–485, and 590 nm for GFP, BFP, and RFP channels, respectively. Bright-field images were used to observe cell morphology. Intensity of fluorescent images was quantified using the UN-SCAN-IT software (Silk Scientific, Orem, UT, USA).

Cells were seeded in 6 well plates in triplicates of either 200, 500 or 1000 cells/well. The cells were incubated for 12 or 16 days in culture medium. At day 12 or 16, cells were fixed 5 min in 3:1 methanol: acetic acid and then stained 15 min in 0.005% crystal violet. Colonies were counted and the percent of cells forming colonies compared to plated cells (100%) were calculated. Morphology of clones and cells in general, were investigated by use of Zeizz Axiovert S100 microscope and the NIS-Elements Documentation software (Nikon).