Figure 3.

Localization of Na⁺ pump α1 and α3 isoforms in primary cultured MA myocytes. (Aa and Ba) Low magnification images of cells crossreacted with mAbs (C464–6B) specific for α1 (Aa) or polyclonal TED antibodies specific for α3 (Ba). The original fluorescent images were filtered by “nearest neighbor” deblurring using CELLscan. (Ab–d and Bb–d) Restored high magnification images of portions of two myocytes. One cell (Ab–d) was crossreacted with polyclonal anti-α1 NASE, and the other (Bb–d), with anti-α3 PcSynA3; both were later treated with DiOC to stain the SR (arrowheads) and mitochondria (arrows) (Ac and Bc). DiOC image Bc was colored red and overlaid on the PcSynA3-labeled image (Bb, green) in Bd; yellow, areas of overlap (note the reticular pattern). Using a 2×2 contingency table (26), we calculated that 5% of the pixels in the image should overlap by chance, whereas we observed 11% overlap. The kappa statistic (where κ = −1.0 to +1.0) can be derived from the contingency table (27); κ > 0 indicates that the overlap is greater than chance. For the data in Bb–d, κ = +0.33. Comparable statistics for Ab–d are: 27% overlap of pixels expected, but only 20% observed, and κ = −0.27. (C) Colocalization of SR Ca²⁺ pump and DiOC staining. The myocyte was labeled with anti-SERCA-2b antibodies (Ca) and then stained with DiOC (Cb); arrowheads in Cb point to SR. (Cc) Overlay of images Ca and Cb; the dense yellow reticular pattern indicates the extensive overlap (19% overlap of SERCA-2b-labeled pixels with DiOC-labeled pixels expected, 39% observed; κ = +0.79). (D) Control cells incubated with preimmune rabbit serum (Da) and then stained with DiOC (Db).