Buff Up on Buffers and Surfactants
Wash buffers are an integral part of immunohistochemical (IHC) procedures. They are used after each incubation step to wash away excess reagent from the specimen and maintain a constant pH by “soaking up” any free hydrogen (H+) ions. This feature helps maintain the morphological characteristics of antibodies and their epitopes, which enables specific binding. The saline and detergent content in buffer solutions also play a role in minimizing background staining.
The two most common buffers in IHC are Tris-Buffered Saline (TBS) and Phosphate-Buffered Saline (PBS). TBS is often used in combination with Tween 20, which reduces nonspecific staining, helps reagents spread evenly across tissue, and may include a preservative (such as sodium azide) to prevent microorganism growth and increase shelf life. TBS is recommended for immunostaining of phosphorylated proteins, as PBS can interfere with “the interaction between phosphorylated proteins and its cognate phosphospecific antibodies.”
This white paper reviews the varying formats of buffers and the advantages of each. To buff up on your buffer knowledge related to IHC, download the complimentary white paper below!
Buffer Tween Slide Comparison
Buffer without Tween20 added. Notice the droplets pooling, which can lead to uneven distribution and uneven staining.
Buffer with Tween20 added. The full slide has even distribution, resulting in even, consistent staining.