Sections stained with the Luxol fast blue-holmes silver nitrate technique show very pale blue myelin and gray axons. The most likely explanation is that

• A. Sections were cut too thin
• B. The fixative was over-fixed
• C. The washing step was insufficient
• D. The stain was expired

Answer: (A)

The key idea is that section thickness directly affects how well a stain can penetrate and reveal its target. Luxol fast blue dissolves into and binds myelin lipids, so when sections are cut too thin there is less tissue mass and less lipid content per field for the dye to interact with. That results in very pale blue staining of the myelin. The Holmes silver nitrate step relies on enough tissue structure to develop the silver properly; if the section is too thin, there isn’t enough material to produce the characteristic dark, black axons, so they appear gray. Together, pale myelin and gray axons point to sections cut too thin, leading to insufficient dye uptake and inadequate silver deposition. For best results, cut sections within the recommended thickness and reprocess if needed.