Fat droplets seen in an Oil Red O stained section are most likely caused by which error?

• A. Pressing on the cover glass to remove air bubbles
• B. Over-staining
• C. Inadequate fixation
• D. Over-dehydration

Answer: (A)

Oil Red O highlights neutral lipids, so what you see should reflect where lipids actually are in the tissue. If the coverslip is pressed hard to remove air bubbles, you mechanically squeeze the tissue and rupture cell membranes, pushing lipid material out and into droplets that pick up the stain. These fat droplets at the surface are artifacts, not true lipid distributions within cells, so this handling step is the likely cause. To avoid it, apply gentler pressure and use appropriate mounting techniques that preserve the integrity of the tissue and its lipids. (Over-staining, inadequate fixation, and over-dehydration each create other issues, but they don’t explain discrete lipid droplets formed by mechanical extrusion.)