Regions of interest were drawn (white dotted outlines) around arrector pili muscles (Figure 1A and 1D), blood vessels (Figure 1B and 1E), and sweat glands (Figure 1C and 1F). Average fluorescence intensity of AS (green), SMA (blue), and TH (red) within the regions of interest was measured using Fiji software after background subtraction and tabulated for each visualized constituent.
For colocalization analyses the method of Jaskolski et al.45 (link) was used, with calculation of the AS/TH colocalization index in the entire image as follows: (1) normalized mean deviation product (nMDP) values for colocalization were tabulated from −1.0 to +1.0 (Figure 2A); (2) the counts corresponding to nMDP values from 0.3 to 1.0 were summed (red or blue boxes in Figure 2A); (3) 0.1 was added, so that the sum of the counts was greater than zero; (4) the log of the number from step (3) was calculated. The formula for the colocalization index therefore was the following.
Mean values (± 1 standard error of the mean) for AS, AS/TH ratios, and AS/SMA ratios for each of the 3 skin constituents and for myocardial NE contents were compared between the Lewy body nOH and non-Lewy body nOH groups by independent-means t-tests conducted on log-transformed data. T-tests were also used to compare mean values for 18F-dopamine-derived radioactivity and clinical and other laboratory parameters between the Lewy body nOH and non-Lewy body nOH groups. Frequencies of values in compared groups were analyzed by Fisher’s exact test. A p value less than 0.05 defined statistical significance.