Table 1.
Unidentified general data of the donors.
Figure 1.
Confocal images of double immunofluorescence staining with Cx43 (green) and N-Cadherin (N-Cad; red) antibodies in rabbit left atrium (LA).
A-C. Representative confocal images of Cx43/N-Cad overlay (A), N-Cad (red; B) and Cx43 (green; C). D-F. Enlarged images from a cropped area of image A including the overlay image (D), N-Cad pixel-by-pixel matched Cx43 (E), and N-Cad non-pixel-by-pixel Cx43 (F). Arrows indicate stellate Cx43 pixels that are in close proximity with the N-Cad pixel but do not co-localize with N-Cad pixel-by-pixel. G. Representative histogram of quantified pixel intensity of Cx43 stained image. H. Representative plot for the mean intensity (blue curve) and standard deviation (red curve) of foreground pixels at different thresholds. I. Representative plot of JT scores corresponding to R-value of four images from two young and two aged rabbit LA. J. Data plot of maximum JT scores of the images from four young and four aged rabbit LA.
Figure 2.
Interaction between Cx43 and N-Cad proteins in human LA.
A. Representative double immunostaining image of Cx43 (green) and N-Cad (red). B-E. Enlarged images from a cropped area of image A including the Cx43/N-Cad overlay image (B), N-Cad (red; C), N-Cad pixel-by-pixel matched Cx43 signals (D), and non-N-Cad co-localized Cx43 (E). Arrows indicate stellate Cx43 pixels are in close proximity with the N-Cad pixel but do not pixel-by-pixel co-localize with N-Cad. F. Immunoblotting images of co-immunoprecipitated N-Cad protein with immunoprecipitated Cx43 proteins. Right column is the immunoprecipitated negative control without primary Cx43 antibody.
Figure 3.
Schematic diagram of the blur algorithm with a dilated N-Cad unit (DNCU) radius of 1 inter-pixel distance (IPD) and 2 IPDs.
A&a. An N-Cad pixel at the center of a DNCU (C&c). Dilation radius equals to 1 IPD and 2 IPD (B&b). D&d. 3 overlapping adjacent DCNUs (R = 1 IPD & 2 IPDs, respectively) containing 3 adjacent N-Cad pixels. E&e. Three Cx43 pixels that are pixel-by-pixel matched with three N-Cad pixels, respectively. F-G & f-g. Two Cx43 pixels that do not co-localize with any N-Cad pixel-by-pixel but are included within the area of one of DCNU. All the DNCU covered Cx43 pixels (E-G & e-g) are defined as Cx43E-E. H&h. Two Cx43 pixels that are located outside of all DCNU covered areas and are therefore categorized as Cx43S-S.
Figure 4.
Determining an optimal radius of DNCU for Cx43E-E and Cx43S-S quantification. A-B & L-M.
Representative Cx43 (green) and N-Cad (red) double immunostaining confocal images (A, L) and grayscale images (B, M) of enlarged single myocytes containing Cx43E-E only (A) or both Cx43E-E and Cx43S-S (L). C-J & N-U. Grayscale images of the enlarged single myocytes with an incrementally increased DNCU radius (1, 5, 7, 30 IPD, respectively) for both N-Cad (left column) and Cx43 (right column). Arrows (U) indicate the inclusion of lateralized Cx43 by an extensively enlarged radius of the DNCU. K & V. Summarized data of quantitative Cx43 values corresponding to a continuously increased radii of DNCU when myocytes present Cx43E-E only (K) or present both Cx43E-E and Cx43S-S (V). The Y-axis value of the first plateau reflects quantitative amount of ID located Cx43E-E (red label) and the difference of the Y-axis value between the first and second plateaus indicates the amount of lateralized Cx43S-S.
Figure 5.
Using N-Cad as an internal housekeeping reference for imaging quantification.
A-B. Confocal images of Cx43 (green) and N-Cad (red) double immunostaining on two LA tissue sections obtained from two young rabbits (Rabbit-1 and Rabbit-2). C. Difference of quantitative total Cx43 raw data between the two confocal images obtained from Rabbit-1 and Rabbit-2 LA. D. A comparable amount of total Cx43 between the two rabbit LA samples when Cx43 raw data was normalized to N-Cad. E. Immunoblotting image of total Cx43 and GAPDH protein expression in Rabbit-1 and Rabbit-2 LA tissue homogenates. F. Comparison of quantitative immuno-stained Cx43 signal intensity with or without N-Cad normalization in aged rabbit LA.
Figure 6.
Comparing results of quantitative Cx43 distribution and abundance using layer-by-layer and maximum projection imaging quantification.
A. Representative sequential confocal images of Cx43 (green) and N-Cad (red) double immuno-stained young rabbit LA from focal layer 1 to layer 6. B. Histogram of quantified Cx43 immuno-stained signals from the maximum projection image. C-D. Summarized data of quantified Cx43E-E and Cx43S-S on young rabbit LA (n = 3; 53 images of each rabbit LA section) using layer-by-layer (B) and maximum projection (C) imaging quantification approaches. D. Pooled data of quantified Cx43E-E and Cx43S-S in young and aged rabbit LA using Layer-by-Layer imaging quantification from Z-stack confocal images (n = 4, 5; *p<0.001).
Figure 7.
Matlab-based algorithm designed to detect interstitial collagen deposition.
A. Representative image of Masson's Trichrome (MT) stained rabbit LA tissue (collagen = blue, myocardium = red). B-D. Representative grayscale images of myocardium (B), unstained white space (C), and blue stained collagen (D). E. Summarized data of quantified interstitial collagen (blue area) in healthy human LA with increasing age (n = 18).