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Fluorescence Microscopy

All reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. Chinese Hamster Ovary (CHO) cells were grown for 2-3 days in $\alpha$-MEM and 10% (v/v) calf serum (Intergen Co., Purchase, NY, USA) on 19 mm cover slips coated with 0.1% (w/v) type I collagen in 0.1 M acetic acid. They were then fixed for 10 min with 2% paraformaldehyde in phosphate-buffered saline (PBS: 140 mM NaCl, 2.6 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 0.9 mM CaCl2, 0.5 mM MgCl2, pH 7.4), permeabilized for 10 min with 0.1% saponin in cytoskeletal stabilization buffer (CSB: 137 mM NaCl, 5 mM KCl, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 4 mM NaHCO3, 2 mM MgCl2, 2 mM EGTA, 5 mM Pipes, 0.1% glucose, pH 6.1) and incubated for 60 min with a primary antibody. After 3 washes of 5 min each in CSB, the cells were incubated for 45 min with 12.5 $\mu$g/ml of a Cy5-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA, USA) and 50 $\mu$g/ml Hoechst 33258 (Molecular Probes, Inc., Eugene, OR USA). The coverslips were washed three more times in CSB before mounting on microscope slides using gelvatol (60 ml of 10 mM Tris, 15 g Airvol 205 (Air Products, Allentown, PA, USA), 30 ml glycerol, 1 g n-propyl gallate). Images of Cy5 and Hoechst 33258 fluorescence were acquired separately using a Zeiss Plan-Neofluar objective (100x, 1.3 NA), and a Photometrics CH 250 cooled charge-coupled device (512 x 382 23 $\mu$m square pixels) mounted on a customized Zeiss Axiovert microscope [22].

Monoclonal antibodies directed against the Golgi protein giantin [23], the lysosomal protein LAMP2 [24], the yeast nucleolar protein NOP4 [25], and tubulin (Sigma) were used as primary antibodies in separate labeling experiments. Working dilutions of antibody stock solutions were obtained by empirically optimizing for low background in the presence of adequate specific signal.

Each slide was scanned for single cells that were spread out on the coverslip (i.e., not rounded up in mitosis). Each such field of view was acquired as a stack of three images in which the focus was changed by 0.237 $\mu$m between each slice. Combined with the 100x objective and 23$\mu$m pixels in the CCD, this slice separation results in nearly cubic 0.23$\mu$m voxels at the object. The image collection is available at, and the number of images per class is included in Table 2.1.


Table 2.1: The number of images in each of the five classes of the CHO data set.
Localization Pattern Number of images
Giantin 77
LAMP2 97
NOP4 33
Tubulin 51
DNA 69
Total 327

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Copyright ©1999 Michael V. Boland