Immunofluorescence ImageReducing the nonspecific binding of secondary antibodies in immunofluorescence workflows is critical to generating high quality data from tissues that action items depend on in the drug discovery process. This is accomplished by using a blocking buffer that is made of normal animal serum containing proteins that bind to reactive sites of the tissue’s antigens, physically blocking the binding of secondary antibodies to those antigens. The primary antibody is still able to recognize and bind to specific antigens on the tissue after blocking, because the blocking buffer binds to antigens on the tissues with less affinity than the primary antibody, outcompeting their overall strength of the hydrogen bonds. Understanding these principles while immunolabeling will generate higher quality and more relevant data sets for researchers. When deciding which type of animal serum to use, the host species of serum used in the blocking buffer should match the host species of the secondary antibody that is used to detect the primary antibody. If the serum used in the blocking buffer was made from the host of the primary antibody, large amounts of nonspecific binding would occur as the secondary antibody would bind to the tissue’s antigens.

Blocking buffers are used during standard immunofluorescence workflows after the tissue has been fixed, sectioned, rehydrated and antigen retrieved (to expose antigens after fixation). In addition to the different types of serums that can be used in the blocking buffer, the buffer the serum is diluted in can change as well. Tris-Buffered Saline (TBS) and Phosphate-Buffered Saline (PBS) can be used to make blocking buffers. However, if you are using phosphorylated antibodies for antigen detection, PBS should not be used as the phosphate group in the buffer can bind to the proteins reducing the overall expression of the signal. While tissue specific optimization is required to determine the concentration of normal animal serum in the blocking buffer, commonly, buffers contain 1% to 6% of normal animal serum in them. Blocking buffers need to be free of impurities as this can reduce expression of the signals.

Rehydrated Kidney Sample

 A properly de- and rehydrated kidney sample labeled for α-Tubulin (green) and nuclei (blue)

Blocking buffers are routinely used at Visikol while performing our highly multiplexed immunofluorescence workflows. After stripping the antibodies off using EasyPlex, the blocking buffer is reapplied to the tissue to repeat the workflow by reducing the nonspecific binding of the secondary antibodies. This method allows Visikol to be agnostic with the antibodies used, and to continue to label the tissue with new antibodies for as long as the tissue remains on the slide.

If you’re interested in learning more about our multiplexing services, please reach out to a member of our team today!


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