Fig. 4. Horizontal and vertical sections through nuclear foci in MEFs (lmna^-/-) transfected with N195K. A transfected nucleus with foci is shown from above (A) or in a side-on section (B) at the position shown by the arrows. The side-on view was created by taking 15 confocal sections 0.25 µm apart in a z-series and assembling them into a three-dimensional image with LaserVox software. (A,B) Staining for transfected lamin A detected with anti-FLAG mAb (red) and endogenous emerin detected with anti-emerin serum (green). (C,D) Controls with identical laser settings, in which cells were stained with anti-FLAG mAb against lamin A (red) and pre-immune rabbit serum (green); in this case, the side-on view was built up from 11 z-series sections instead of 15. (E-G) Confocal sections (top to bottom) of a nucleus containing five foci. Two of these foci (asterisks in E) appear to be attached to the nuclear rim, where emerin is concentrated. Of the remaining three central foci visible in F, two appear to be attached to the upper surface (E) and one to the lower surface (arrow in G). The inset labelled LAP2 shows a double label with rabbit anti-FLAG for lamin A (green, ALEXA488 anti-rabbit Ig) and a commercial mAb against LAP2 (red; ALEXA546 anti-mouse Ig). The inset labelled LAMB shows a double label with monoclonal anti-FLAG for lamin A (green; FITC anti-mouse Ig) and a commercial goat antibody against lamin B (red; TRITC anti-goat Ig). Bars, 10 µm.

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