Wash buffers remove unbound antibodies and excess reagents from the tissue sections before adding new reagents as part of a sequential IHC staining protocol. Wash buffers may also mix secondary antibodies, link reagents, and labels in the staining process. Wash buffers may contain detergents that aid in the sheeting action for stringent washes. It is essential that the wash buffer is compatible with other staining reagents used and that it does a thorough job. Incomplete rinse steps, as well as ineffective wash buffers, will contribute to nonspecific background staining. It is essential when preparing buffers that:
- The pH is optimal
- It is a pure buffer and not a mixture of buffers
- All the ingredients are fresh and not outdated
- Proper storage is adhered to
Accurate storage of wash buffers is important to preserve stability and consistency when using them. Concentrated wash buffers are typically stored at 2–8°C, while working wash buffers are most commonly stored at 20°C. Some concentrated buffer solutions diluted and mixed before use may form crystals if stored in the refrigerator. These should be brought to room temperature while stirring to dissolve the salts before mixing the components and checking the pH.
The most commonly used wash buffers in IHC methods are tris-buffered saline (TBS) and phosphate-buffered saline (PBS).
