Figure 2.

Laser scanning confocal microscopy of transcription sites, their segmentation, and spatial relationship with the nuclear lamina in permeabilized 3T3 cells. (A) Nucleolar transcription sites are encircled (yellow dashed lines). A cluster of transcription sites is indicated by red arrows. Regions with no transcription activity were indicated with green arrows. (B) A segmentation algorithm is applied to the image in A. Every site counted is contoured with a green line. (C) Higher magnification of a portion of A. (D) Higher magnification of a portion of B. (E–G) Double labeling of transcription sites and nuclear lamina. (E) Transcription sites are stained with FITC (green). (F) Nuclear lamina is detected with antilamin B antibodies and Texas red–conjugated secondary antibodies (red). (G) Merged image of transcription sites and nuclear lamina shows that transcription is occurring inside the region confined by nuclear lamina, and transcription sites are not associated with lamin B either on the lamina or with internal lamin B positive spots. (H–J) A portion of the image, indicated with a box in the merged image, is further enlarged. All images are 0.5-μm midplane optical sections.