Figure 2

Islet NCAM and TH axon image analysis by confocal microscopy and ImageJ in control donors. Frozen sections (40 µm thick) were fixed and stained for neural cell adhesion marker (NCAM), tyrosine hydroxylase (TH) and glucagon (α-cells, see Fig. 3a) to define total and sympathetic innervation and islets, respectively, as described in Methods. Islets were contoured on maximum intensity projects as demonstrated in (a). (a) The z-stack confocal images of 40 µm thick sections stained for NCAM and TH were converted into MIP images (A) NCAM in red, (B) TH in green and (C) merged image representing colocalization NCAM and TH. The images D, E, F are the corresponding threshold images to (A), (B) and (C) respectively. These raw threshold images were quantified for % density per unit area for NCAM and TH within islets. (b) The thresholded images were quantified for NCAM and TH density (%). (c) The thresholded images were quantified for TH/NCAM overlap (%). Data are scatterplots with bars representing mean ± SD for 7 islets/donor in 7 control donors, aged 9–21.8 years old. See also Supplementary Fig. S2.