Which reagent is used to remove the red dye from collagen in the Masson Trichrome protocol?

• A. Acetic acid
• B. Phosphotungstic acid
• C. Calcium chloride
• D. Ethanol

Answer: (B)

Phosphotungstic acid acts as the differentiating step in Masson’s trichrome. After the tissue is stained with a red dye to color muscle and cytoplasm, this reagent selectively removes the red from collagen while leaving other components ready to take up the blue counterstain. That selective decolorization is what lets collagen bind the blue dye (often aniline blue) in the next step, producing blue collagen while muscle remains red and nuclei stay dark. Other reagents in the protocol—such as ethanol, which is for dehydration, or acetic acid and calcium chloride, which have different roles in staining or masking—do not perform this targeted removal of the red dye from collagen.