Sections stained with PTAH show blue glial fibers, but the neurons are unstained. One possible cause is that the sections were washed too long before clearing and mounting.

• A. The sections were washed too long before clearing and mounting
• B. The sections were stained too long in PTAH
• C. The sections were fixed in formalin
• D. The staining solution was too old

Answer: (A)

In PTAH staining, the blue color marks glial fibers because the stain binds selectively to glial fibrils, while neurons take up a different color from the stain or counterstain. If the sections are washed too long before clearing and mounting, the soluble dye can diffuse out of the tissue. Neurons, which depend more on the cytoplasmic staining for visibility, are more susceptible to being washed clean, so they can become unstained. In contrast, glial fibers bind the stain more tenaciously and can retain their blue color despite extended washes. So, excessive washing before the clearing step can selectively remove neuronal stain while leaving glial staining intact, making this a plausible cause for the observed pattern.