What mounting media correspond to each counter stain in immunohistochemistry?

• A. HRP-DAB Neutral; AEC Aqueous mount; AP- BCIP/NBT Neutral; AP- Fast Red Aqueous
• B. HRP-DAB Aqueous; AEC Neutral; AP- BCIP/NBT Aqueous; AP- Fast Red Neutral
• C. HRP-DAB Neutral; AEC Neutral; AP- BCIP/NBT Neutral; AP- Fast Red Neutral
• D. HRP-DAB Neutral; AEC Aqueous mount; AP- BCIP/NBT Aqueous; AP- Fast Red Neutral

Answer: (A)

Mounting media are chosen to preserve the color and integrity of both the chromogen and any counterstain, based on how soluble or unstable each stain is in processing solvents. Hydrophilic, water-soluble reagents need an aqueous mounting medium to prevent washing away, while stable chromogens can stay in a neutral or non-aqueous mount without fading.

For HRP with DAB, the brown product is relatively stable and is best preserved with a neutral mounting medium that doesn’t introduce pH changes that could affect DAB’s color.

For AEC, the red chromogen is water-soluble, so it must be mounted with an aqueous mounting medium to prevent dissolution and loss of color.

For alkaline phosphatase with BCIP/NBT, the precipitate is best preserved under neutral conditions, so a neutral mounting medium is appropriate.

For alkaline phosphatase with Fast Red, this color product is more compatible with aqueous mounting to maintain its vibrant red hue without solubilization during dehydration steps.

In short, water-soluble or aqueous-sensitive stains require an aqueous mount, while stable chromogens like DAB are best paired with a neutral mounting medium.