In immunohistochemistry (IHC), when using alkaline phosphatase (AP)-conjugated detection systems, it is important to address endogenous AP activity that can cause nonspecific background staining, similar to endogenous peroxidase in HRP-based systems.
Why Quench Endogenous AP?
Endogenous alkaline phosphatase is naturally present in various tissues such as kidney, intestine, osteoblasts, lymphoid tissues, and placenta. It is often more prominent in frozen tissue sections. If not quenched, endogenous AP can react with AP substrates like BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium), producing unwanted blue/purple background staining.
Methods for Quenching Endogenous AP
- Levamisole Addition: The most common method to inhibit endogenous AP is to include levamisole, a specific AP inhibitor, in the substrate solution. Levamisole at around 1 mM concentration selectively inhibits endogenous AP without affecting the activity of the alkaline phosphatase conjugated to the detection antibody. Many commercial AP chromogen kits include levamisole in their substrate mixes.
- Heat-Induced Epitope Retrieval (HIER): Endogenous AP activity can also be reduced or destroyed by heat treatment during antigen retrieval (e.g., boiling in citrate or EDTA buffers), which denatures endogenous enzymes.
Testing for Endogenous AP
Before staining, tissues can be tested for endogenous AP by incubating sections with BCIP/NBT substrate alone. If blue staining appears, blocking is necessary.
Buffer Considerations
Avoid PBS with AP detection: Unlike HRP protocols, phosphate-buffered saline (PBS) is avoided during AP staining because phosphate ions inhibit alkaline phosphatase activity. Instead, Tris-buffered saline (TBS) is recommended for washing and reagent dilution.
Summary of AP Quenching Steps
- Prepare tissue sections (deparaffinize, rehydrate, antigen retrieval).
- Test for endogenous AP activity if uncertain.
- Include levamisole in the AP substrate solution to inhibit endogenous AP.
- Use TBS instead of PBS throughout the staining procedure.
- Proceed with AP-conjugated antibody incubation and chromogen development.
