Fig 2: 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.

Fig 3: Quantitative analysis of the axonal diameter.(A,C,E,G) Confocal images of TUBB3 immunostaining (white) of skin biopsies from (A) male proximal, (C) male distal, (E) female proximal, and (G) female distal leg. (B,D,F,H) Graphs depict axon diameter (µm) in (B) male proximal, (D) male distal, (F) female proximal, and (H) female distal skin biopsies (3 images per individual, = 20 axons per image). (I–L) Axon diameter distribution for the (I) male proximal, (J) male distal, (K) female proximal, and (L) female distal sample sets. Diameter spans are defined as follows: < 2.00 µm, 2.01 = … < 3.00 µm, = 3.01 µm. Scale bars in (A,C,E,G) are 5 µm. Points in graphs (B,D,F,H) symbolize one individual. Data represent mean ± SEM. (B,D,F,H) Kruskal–Wallis test with Dunn’s multiple comparisons test. (I-L) Two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.