Fig 2: Display of experimental procedure and quantification of sensory nerve fiber density. (A-1) Schematics of skin biopsy location: proximal (thigh) and distal (ankle) region of the leg. (A-2) Skin sections immunostained for cytoskeletal proteins. Created with BioRender.com. (A-3) Confocal image of a skin biopsy section stained for type III ß-tubulin (TUBB3, red) and DAPI (nuclear stain, blue). (B) Images for immunohistochemical staining for PGP9.5 of two individuals of the youngest (20s, left) and the oldest (70s, right) age group. Higher magnification of boxed area shows a single fiber. (C–F) Quantification of IENFD for (C) male proximal, (D) male distal, (E) female proximal, and (F) female distal sample sets. One data point in (C–F) represents the IENFD value of one individual. Data represent mean ± SEM. Scale bars in (A-3) and (B) are 50 µm.

Fig 3: Quantitative immunostaining of human skin sections for TUBB3. (A,B,F,G) Confocal images of human skin biopsies immunostained for TUBB3 (white) with nuclear stain (Hoechst, blue) taken from individuals of the four age groups (20s, 40s, 60s, and 70s) from (A) male proximal, (B) male distal, (F) female proximal, and (G) female distal samples. (C,D,H,I) Quantification of TUBB3 gray values in sensory axons (x-fold normalized to average of 20s group) (3 images per individual, = 20 axons per image, (C) n = 51 individuals in proximal male, (D) n = 27 in distal male, (H) n = 21 in proximal female, (I) n = 20 in distal female data). (E,J) Male and female proximal and distal sample sets were compared via the proximal/distal TUBB3 expression ratio. Scale bars in (A,B,F,G) are 10 µm. Points in graphs (C,D,H,I) represent on individuals. Data represent mean ± SEM. Kruskal–Wallis test with Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.