Which antibody is best for determining when tissue has been overfixed in immunohistochemistry?

• A. Vimentin
• B. CD20
• C. Cytokeratin
• D. GFAP

Answer: (A)

Overfixation leads to excessive cross-linking that masks epitopes and reduces antigen accessibility. To gauge fixation quality, you want an antigen whose epitope is particularly sensitive to that cross-linking so its staining drops when fixation is too long or too harsh.

Vimentin fits this role well. It’s a cytoskeletal protein widely expressed in many cell types, and its epitopes can be notably affected by overfixation. If tissue has been overfixed, vimentin immunostaining is more likely to be diminished or lost, making it a practical indicator of overfixation.

In contrast, CD20, cytokeratin, and GFAP are important markers but their staining can be influenced by tissue type and cell presence, and they’re not as reliable as a universal gauge of fixation quality across different specimens. Hence, vimentin serves as the best choice for assessing potential overfixation.