Following hybridization, slides were evaluated using fluorescence microscopy with SpectrumGold, SpectrumOrange™, SpectrumRed, SpectrumGreen, and SpectrumAqua fluorescence filters (Vysis/Abbott Molecular, Inc., Des Plaines, IL).
Gross analysis of tumors and dissected femurs (soft tissue removed) was performed using a Leica MZFLIII dissecting microscope with bright field and GFP fluorescence filters [λexcitation = (480 ± 20) nm; λemission = 510 nm barrier; Leica, Deerfield, IL].
It was used to acquire the fluorescence, filter the expressed and non-expressed spots, align the spots, quantify their intensity and finally export GenePix report (GPR) files containing spot intensity raw data.
Epifluorescent images were visualized on a Leica microscope (47-nm cyan fluorescent protein, fluorescence filter set 47 (EM BP 436/20, BS FT 455 and EM BP 480/40); Leica Microsystems, North Ryde, 2113 Australia).
Depending upon the assay, one of the following three fluorescent filters was used: FITC HYQ fluorescence filter (460-500 nm); TX RED HYQ fluorescent filter (532-587 nm); and ultraviolet filter (325-375 nm).
Although this illumination method can be used for scattered light observations as well, we use this method mainly with monochromatic illumination and fluorescence filters recording samples stained with fluorescent markers.
To effectively separate the images from different fluorescent dyes, it was necessary to set up an appropriate set of fluorescence filters.
The fluorescent transformed seeds were selected under an inverse microscope equipped with a DsRed fluorescence filter (Axiovert 200M; Zeiss AG, Germany) and put on soil to grow the next generation.
Cells were visualized with a microscope equipped with fluorescence filter.
The images were viewed under the fluorescence microscope (×1,000 amplification) with a green fluorescence filter.
