Frequently Asked Questions - Immunolabeling

How do I know which type of immunolabeling procedure should be employed to properly label my target antigen of interest?
The following is a general guideline for the type of immuno-labeling procedure utilized, depending upon the location of the target protein:
  • Pre-Embedding Immunolabeling: Pre-Embedding Immunolabeling is used primarily for the detection of cell-surface associated antigens, as well as external envelope antigens of budding or extracellular virus particles.
  • Negative Stain Immunolabeling: Negative Stain Immunolabeling is performed on specimens found in concentrated suspensions, such as virus particles, cell fragments, macromolecules, and bacterial specimens. Both internal and surface-related target proteins on minute specimens can be immunolabeled using this procedure.
  • Post-Embedding Immunolabeling: Post-embedding labeling is used exclusively for the immunolabeling of intracellular proteins found within cells and tissues.
How do I prepare my samples for immunolabeling procedures?

For pre-embedding immunolabeling, the cells are exposed to both the primary antibody and secondary antibody in situ, before normal fixation procedures. Because we work with the cells live or in situ, the cell line would be sent to ABI fresh in their normal cell culture media.

Negative stain immunolabeling is performed on fresh specimens in suspension before chemical fixation is employed, thus allowing for the antigenicity of the sample to be intensified. The specimens would be sent to ABI either fresh or frozen and would be handled as a "live" sample.

Specimens to be examined by negative staining techniques may have to be concentrated before immunolabeling techniques.

For post-embedding labeling procedures, immunolabeling is carried out on cells and tissues that have been fixed and embedded in a plastic resin. An embedded block containing the specimen is ultra-thin sectioned, thus allowing access to antigens as they become exposed on the surface of the sections.

Following this procedure, the cells and/or tissue must be fixed using a chemical fixative before shipment to ABI. Extensive testing of the fixatives and chemicals used during processing is performed by both ABI and the investigator to maximize the probability of successful immunolabeling.

Who provides the primary and secondary antibodies for immunolabeling services? How much volume of antibody should I provide?

For immunolabeling experiments, regardless of what type of procedure is to be employed, it is the responsibility of the investigator to provide the primary antibody, as well as information regarding the recommended concentrations and/or dilutions to be used for the experiment.

ABI will provide the secondary antibody or conjugate used for immunolabeling the specimens.

We strongly recommend that the investigator provide at least 200 microliters of the antibody to be utilized for immunolabeling procedures, although the total volume needed does depend on both the type of service to be performed and the strength of the primary antibody being used.

Due to some of the complexities that are involved with successfully performing immunolabeling techniques, we ask that before sending in your samples for immunolabeling services, you call and discuss your project with us directly over the telephone.