 Methods & Techniques For Histologists and Immunohistochemists |
Cell Biology Applications of Fluorescence Microscopy
Stephen Rogers
Manager, Microscopy Suite, Light and Confocal Microscopy
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 N. Mathews, Urbana, IL 61801
Specimen Preparation
Blocking
When using antibodies to stain specimens, it is often necessary to block the sample to minimize non-specific binding. Non-specific binding may occur for several reasons: un reacted aldehydes within the preparation may crosslink antibodies to inappropriate structures (especially with glutaraldehyde); highly charged or very hydrophobic structures within the samples may 'trap' antibodies; or, if using polyclonal antibodies, low affinity IgGs may bind speciously to structures that you are not interested in. These potential pitfalls may be prevented by treating the specimens with a protein solution that will compete for non-specific binding sites prior to staining with antibodies. Commonly used blocking agents are bovine serum albumin, casein (or a solution of non-fat dry milk), gelatin, or normal serum obtained from the species of animal in which the secondary antibodies are made. Typically, the protein solutions are used at concentrations of 1 to 10% in buffer and the samples are treated after permeabilization for 10 to 30 minutes. It is also advisable to include a small amount of detergent (if the sample is to be permeabilized) to compete for hydrophobic interactions with the antibodies. In this case, low (around 0.1%) concentrations of Triton X-100 should be used. In my experience, normal serum has proven to be the blocking agent of choice.