378
JINGLY FUNG et al.
6. Wash slides in 0.1% Tween 20/4xSSC at room temperature for 2 min.
Then, 80 I-li of blocking reagent (vial 2, SKY kit) are applied to each
slide. Each slide is covered with a plastic coverslip, and incubated
at 37°C for 45 min.
7. After careful removal of the coverslip, 80 I-ll of buffer I (vial 3, SKY kit,
containing avidin Cy5 and mouse-anti-digoxin) are added to each slide
and slides are recovered with a plastic coverslip. Incubate the slides at
37°C for 45 min.
8. After careful removal of the coverslip, wash the slides three times in
0.1% Tween 20/4xSSC at room temperature for 5 min on the shaker.
Then, 80 I-ll of buffer II (vial 4, SKY kit, containing goat anti-mouseantibody conjugated to Cy5.5) are applied to each slide and slides are
recovered with a plastic coverslip. Slides are incubated at 37°C for 45
min.
9. After careful removal of the coverslip, slides are washed three time in
0.1% Tween 20/4xSSC at room temperature for 5 min each on a shaking platform, and finally, the slides are mounted in 10 I-ll of 4, 6-diamidino-2-phenylindole (DAPI, vial 5, SKY kit).
$ubprotocol 4
Image acquisition and analysis
Images are acquired with the SD200 SpectraCube Spectral Imaging system
(AS., Inc.). The SD200 imaging system attached to a Nikon E600 microscope consists of an optical head (Sagnac interferometer) coupled to a
multi-line CCD camera (Hamamatsu). The image data is stored in a Pentium 586/300 MHz computer and analyzed by the system software. The
multiple band pass filter set used for fluorochrome excitation is custom-designed (SKY-I, Chroma Technology) to provide broad emission
bands (giving a fractional spectral reading from -450 to -850 nm). Using
a Xenon light source, the spectral image is generated by acquiring 80-130
interferometric frames per object. The sample emission spectra are measured in the visible and near infrared spectrum simultaneously at all
points in the microscopic image. The CCD camera captures images at discrete interferometric steps that are stored as an image stack in the computer. Next, each interferogram is Fourier transformed resulting in a
fluorescence spectrum for each pixel of the image. DAPI images are recorded using a DAPI specific optical filter set.
JINGLY FUNG et al.
6. Wash slides in 0.1% Tween 20/4xSSC at room temperature for 2 min.
Then, 80 I-li of blocking reagent (vial 2, SKY kit) are applied to each
slide. Each slide is covered with a plastic coverslip, and incubated
at 37°C for 45 min.
7. After careful removal of the coverslip, 80 I-ll of buffer I (vial 3, SKY kit,
containing avidin Cy5 and mouse-anti-digoxin) are added to each slide
and slides are recovered with a plastic coverslip. Incubate the slides at
37°C for 45 min.
8. After careful removal of the coverslip, wash the slides three times in
0.1% Tween 20/4xSSC at room temperature for 5 min on the shaker.
Then, 80 I-ll of buffer II (vial 4, SKY kit, containing goat anti-mouseantibody conjugated to Cy5.5) are applied to each slide and slides are
recovered with a plastic coverslip. Slides are incubated at 37°C for 45
min.
9. After careful removal of the coverslip, slides are washed three time in
0.1% Tween 20/4xSSC at room temperature for 5 min each on a shaking platform, and finally, the slides are mounted in 10 I-ll of 4, 6-diamidino-2-phenylindole (DAPI, vial 5, SKY kit).
$ubprotocol 4
Image acquisition and analysis
Images are acquired with the SD200 SpectraCube Spectral Imaging system
(AS., Inc.). The SD200 imaging system attached to a Nikon E600 microscope consists of an optical head (Sagnac interferometer) coupled to a
multi-line CCD camera (Hamamatsu). The image data is stored in a Pentium 586/300 MHz computer and analyzed by the system software. The
multiple band pass filter set used for fluorochrome excitation is custom-designed (SKY-I, Chroma Technology) to provide broad emission
bands (giving a fractional spectral reading from -450 to -850 nm). Using
a Xenon light source, the spectral image is generated by acquiring 80-130
interferometric frames per object. The sample emission spectra are measured in the visible and near infrared spectrum simultaneously at all
points in the microscopic image. The CCD camera captures images at discrete interferometric steps that are stored as an image stack in the computer. Next, each interferogram is Fourier transformed resulting in a
fluorescence spectrum for each pixel of the image. DAPI images are recorded using a DAPI specific optical filter set.