Figure 3.

Formalin-fixed normal Achilles tendon stained with picrosirius red (PSR). (A) Polarized view of representative entire longitudinal Achilles tendon section. (B–D) Magnifications of approximately the same region as the green box in panel A at three different orientations: 0°(B), 45°(C), and 90°(D). Collagen fibers showed different birefringence colors depending on the tendon orientation. Overall, collagen was displayed predominantly as a parallel arrangement of thick, yellowish-orange birefringent fibers. At 45°(C), the collagen fibers appeared uniformly bright yellow. At 0°(B) and 90°(D), a striped pattern was seen, with some fibers at extinction (arrows). White arrowheads in D show some greenish collagen fibers colocalized with thick, yellowish-orange fibers. (E–G) PSR images acquired in the same regions of interest as panels B–D were quantitatively compared for the percentage of total collagen using the Count & Measure module of the cellSens software. Blue indicates background; the sum of red and yellow is total collagen, represented as red in the quantitation panel. Tendon overview image (A) was obtained with a 4× magnification lens (scale bar: 500 µm). Higher magnification images within the boxes (B–F) were obtained with a 20× magnification lens (scale bar: 50 µm).