Figure 1

Immunolocalization of mABs SMI-31, SMI-32, anti-BrdU, and pAB anti-GFAP. A. A micrograph showing a single, confocal image plane of SH-SY5Y cells labeled with SMI-31 mAB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by the TO-PRO-3 DNA probe (blue). The green SMI-31 mAB labeling appears as white dots within the blue nuclei, and as filamentous green structures in the cytoplasm. B. A single image plane of SH-SY5Y cells grown on glass coverslips and labeled with RT97 mAB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by TO-PRO-3 probe (blue). RT97 mAB labeling appears as white dots in nuclei and as filamentous or clumpy green structures in the cytoplasm C. A confocal projection of SH-SY5Y cells grown on glass coverslips and labeled with SMI-32 mAB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by TO-PRO-3 probe (blue). SMI-32 mAB did not label nuclei but did reveal filamentous green structures in the cytoplasm. D. A confocal projection of F98 rat glioma cells grown on glass coverslips and labeled with SMI-31 mAB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by TO-PRO-3 probe (blue). SMI-31 mAB labeling appears as white dots in nuclei and as filamentous green structures in the cytoplasm. Labeling in these cells was so intense that the nuclei appeared completely full of the labeled epitope. E. A confocal projection of F98 rat glioma cells grown on glass coverslips and labeled with RT97 mAB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by TO-PRO-3 probe (blue). SMI-31 mAB labeling appears as white dots in nuclei. RT97 staining of nuclear and cytoplasmic structures in F98 rat glioma cells was similar but less intense than seen with SMI-31 on F98 cells. F. A confocal projection of F98 cells grown on glass coverslips and labeled with anti-GFAP polyclonal AB followed by an Alexa-Fluor-488 conjugated secondary antibody (green). Nuclei were revealed by TO-PRO-3 probe (blue). The anti-GFAP revealed the expected filamentous green structures in the cytoplasm but did not label structures in nuclei. The white areas that look as if they are in the nucleus are actually green staining in the cytoplasm below (seen through) the blue nucleus. G, H, I. Confocal projections of SH-SY5Y cells showing nuclei labeled with TO-PRO-3 (blue) and anti-BrdU (green, white when co-localized with blue). These panels show nuclei of cells in culture for 1, 3, and 6 days, respectively. Labeling intensity appeared to decline as the culture became confluent, suggesting a corresponding decline in DNA synthesis and cells exiting the cell cycle. See also Figure 4. *the calibration bar in each panel represents 5 μm.