Previous: Step 1, Antigen Retrieval
Step two in our Better IHC series covers primary antibody diluent.
Good IHC protocols provide the proper conditions for a specific binding event to occur. It is therefore essential that you calibrate the ionic strength and pH of the antibody diluent to the specific antigen epitope interaction you are trying to detect. TBST is commonly used for this purpose. It is isotonic to normal saline, in buffered around physiological pH.
These conditions mimic the environment that antibodies and antigens encounter during the natural course of an immune system interaction. As a result, TBST works well for many antibodies, such as this antibody for Phospho-EGF Receptor. But it doesn't work for all antibodies.
