Cfi plan apo 10

KR12-TAMRA (1, 5 or 25 μg) was injected intravenously into the chicken egg, and then tissue distribution of KR12-TAMRA was investigated by red fluorescence of KR12-TAMRA using a fluorescent stereomicroscope. RITC (Rhodamine-B isothiocyanate, 25 μg) was used as a control. In addition, the CAM tumor was fixed overnight with 4% paraformaldehyde at 4 °C, and its tumor (after washing with ice-cold PBS) was treated with 99.8% methanol for 30 min at −80 °C and washed in ice-cold PBS again. The tumor was incubated in a 20% sucrose solution overnight at 4 °C. Thin sections (sliced with 30 µm in thickness by the cryomicrotome) were prepared from the tumor and observed by using a confocal laser microscope (Nikon First-Scan Confocal Microscope A1R) which was equipped with a 10× lens (CFI Plan Apo 10, Nikon, Tokyo, Japan). The wavelength of excitation (Exc) and fluorescence emission (Emis) was GFP, Exc at 488 nm and Emis at 500–550 nm; TAMRA, Exc at 561 nm and Emis at 570–620 nm.

The spectral confocal laser scanning microscopy (SCLSM) analysis of the stained samples was performed at 25 °C using the SCLSM system (Digital Eclipse C1si; Nikon) equipped with CFI S Fluor 4×, CFI Plan Apo10×, 20×, 40×, and VC60×H lenses and the EZ-C1 3.40 software (Nikon). Fluorescence was excited with the 408-nm line of a blue diode laser, the 488-nm line of a solid laser, and the 640-nm line of a diode laser. The emission spectra in the ranges of 460–470 nm, 500–650 nm, and 640–750 nm with 5 nm bandwidths were recorded for detecting the cell wall, TPI and lipid, and Alexa Fluor 647, respectively, with TPI and lipid simultaneously detected in the rage of 500–650 nm. Reference samples were prepared by dissolving commercial trans-polyisoprene in chloroform at a final concentration of 1 mg/mL, and thin films were prepared on glass slides. The films were stained with Nile red, and fluorescence spectra from each of 50 locations (regions of interest) were measured and averaged. The fluorescence spectra of the stained samples were obtained from 10 to 25 locations and averaged. Images were acquired and averaged from five successive scans to improve the signal-to-noise ratio. Image processing, including spectral unmixing, was performed using EZ-C1 3.40 software and Adobe Photoshop CS4.