Which of the following is a common cause of uneven H&E staining in processed tissue?

• A. Water contamination of the clearing agent
• B. Over-oxidation of tissue
• C. Excess mounting medium
• D. Over-fixation

Answer: (A)

Uneven H&E staining happens when stain uptake varies across the tissue, which often comes from problems during clearing and subsequent steps. If the clearing agent is contaminated with water, it can’t properly replace the alcohol and paraffin, leaving residual moisture in parts of the tissue. That moisture disrupts uniform clearing and infiltration, so different areas of the section take up hematoxylin and eosin unevenly, creating patchy or streaky staining. Clearing agents must be dry and fully compatible with paraffin to ensure consistent dye penetration. Other factors like over-oxidation, over-fixation, or excess mounting medium can affect overall color intensity or appearance, but they’re less likely to cause the distinct patchiness that results from moisture in the clearing step.