In the Verhoeff-Van Gieson stain, collagen and muscle are not differentially stained and the elastic fibers do not contrast well with the background. Which of the following is a documented cause?

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Verhoeff-Van Gieson relies on a stable Van Gieson counterstain to differentiate collagen from muscle after the elastic fibers have been stained black. If the Van Gieson counterstain is washed out during processing, the pink/red collagen and yellow muscle colors are lost or severely diminished. Without that color contrast, collagen and muscle can appear similar in shade, and the elastic fibers no longer stand out against the background, giving a washed-out, non-differentiated appearance. This exact staining pitfall is well documented, and maintaining the counterstain intact is essential for proper differentiation. Using fresh Van Gieson, avoiding excessive rinsing, and following correct timing help prevent this issue. The other scenarios described are not typical causes of this particular staining failure.