A. Antigen Retrieval (US Patent Nos. 5,244,787 and 5,578,452 and their foreign equivalents) is a technique used to unmask antigens that have been covered or altered by fixation. The most common method involves routinely heating paraffin sections in a retrieval solution, then cooling, rinsing, and proceeding with routing IHC staining.
Q. How does Antigen Retrieval work?
A. The effectiveness of Antigen Retrieval may be due to a combination of mechanisms. The Antigen Retrieval technique breaks the formalin induced cross-linking bonds between epitopes and unrelated proteins, allowing better penetration of antibody and accessibility of epitopes. Some proteins may also be denatured or renatured, thus exposing the epitope.
Q. What are the benefits of doing Antigen Retrieval?
A. Antigen Retrieval can produce markedly improved staining for a wide range of monoclonal and polyclonal antibodies. It helps overcome false negative staining of over-fixed tissue, expand the range of antibodies useful for routinely processed tissue, and increases the usefulness of archival materials for retrospective studies. It may also help reduce background staining, and allows one to titrate the antibody to a higher dilution.
Q. Does every antibody need Antigen Retrieval?
A. Antigen Retrieval has in many cases proven to be superior to enzyme pre-treatment, but in some cases it is not. If the manufacturer has not previously tested with various retrieval methods, one should test and compare. In cases where enzyme pre-treatment is needed and when the tissue is optimally fixed, Antigen Retrieval may not be necessary.
Q. What kind of equipment will I need to do Antigen Retrieval?
A. The most basic equipment required is a microwave oven (such as our very own EZ Retriever™ ), preferably one with a rotating tray, a microwave-safe staining dish and a slide holder (BioGenex Catalog Number MW001-SU), a cylinder to measure solutions, and a few paper towels to place under the container. Also required are antigen retrieval solutions needed for the particular antibody being used.
Q. How does one do Antigen Retrieval?
A. There are several methods for performing Antigen Retrieval, but lack of standardization can lead to problems. When various methods and equipment are used with different protocols, one may see variable results from day to day and from lab to lab. This is likely to result in discrepancies in staining.
BioGenex has developed two procedures that improve standardization and provide reliable immunostaining:
The Standard Microwave Protocol - Involves the use of a microwave, which allows for flexibility, cost-effectiveness, and reproducibility.
The Microwave Pressure Cooker Protocol - Involves using a plastic pressure cooker in addition to a microwave.
Q. What factors will affect my results?
The following variables will affect the quality of results:
The temperature of the retrieval solution. It should be brought to room temperature before it is used. Cold solution will take longer to boil and if the time is not compensated, retrieval may not be completed. Rapid boiling is one of the most important criteria.
The pH and molarity of the solution.
The number of slides in the holder. Fewer slides will take longer to bring to an initial boil.
Wattage of the microwave. This is more of a problem with older microwave ovens, since the wattage may be low or unknown.
The size and color of the container which holds the retrieval solutions. Containers that are more vertical, such as Coplin jars, allow the solution to easily boil over and the tissues to dry out. If the container is colored or opaque it may be difficult to see when the solution comes to an initial boil.
Q. Do I need a license to practice BioGenex patented method of microwave Antigen Retrieval?
A. BioGenex will grant a license waiver for non-commercial testing. Academic research requires no license. Contact BioGenex for more information on licenses for commercial testing.
Q. Why doesn't heating destroy my protein?
A. Significant denaturalization of proteins does occur at temperatures between 70oC to 90oC, but formalin-fixed proteins are more heat stable. Cross linkage of protein produced by formalin fixation seems to protect the epitopes.
Q. What happens if my solution boils over?
A. The tissue can fall off the slide from over-vigorous boiling action and/or due to the solution boiling over. The ideal condition is to place the slides in a proper container, with a loose fitting lid, and ensure that the temperature is reduced to a simmer to prevent the harsh boiling action on the tissue. If the solution does boil out, it should be refilled with additional solution or distilled water.
Q. What causes the solution to boil away when using Coplin jars for Antigen Retrieval?
A. Generally, the use of Coplin jars is not recommended. We recommend a microwave slide holder container (BioGenex Catalog Numbers MW001-SU and MW001-HB). These containers are larger, and are microwave safe. However, if a Coplin jar is used, it is really important to closely watch the first boiling step to ensure that the solution does not boil over. Then lower the power so the solution simmers. Also for consistency, make sure that the Coplin jar is placed in the same position within the microwave each time.
Q. How can I make sure the tissue will remain on the slides during Antigen Retrieval, and what causes some to fall off?
A. The most important prevention is to have the tissue mounted on positively charged slides, or having poly-L-Lysine coated on the slides. When this criterion is met, one must consider the tissue. Often, tissues that have a large amount of collagen or fatty substances have a tendency to fall off. A modified method of retrieval can be used to aid in their adherence on the slide. Bring the solution to a boil in the microwave first, and then add the slides. Turn the temperature to low and let the tissues simmer for a reduced time of 3 to 5 minutes. There is a possibility that the reduced time may cause incomplete Antigen Retrieval. Remove the slides from the microwave and let them cool naturally for 20 to 30 minutes. For these tissues, it also helps to use deionized water in the floatation process when they are initially placed on the positively charged slides.
Q. Can we speed up the cooling process by putting the slides in cold water?
A. The best procedure is to allow the slides to cool naturally for 20 to 30 minutes. This is part of the process of bond breaking that unmasks the epitope. We find stronger staining when the slides are cooled to room temperature in a natural way.
Q. What is the Microwave Pressure Cooker Protocol, and what are its advantages?
A. The Microwave Pressure Cooker method is a procedure utilizing the microwave with a pressure cooker as a container for the slides and the solution. The pressure cooker sold by BioGenex is especially designed for use in a microwave oven. Its advantages include the speed of treatment, the reproducibility of results with large batches of slides, the ability to use metal slide racks, and economy of time and equipment costs.
Q. What is the cause of occasional artifacts in my negative control using the Microwave Cooker Protocol?
A. This may be due to the concentration of negative control sera used. Sensitivity may increase using the Microwave Pressure Cooker protocol.
Q. What causes non-specific staining in my negative control?
A. The problem with the negative control showing non-specific staining has most often to do with the antigen exposing endogenous biotin. Antigen Retrieval sometimes makes the tissue act like a frozen tissue, and the detection system may be too strong, or the antibody too concentrated. Further titration may be necessary. Doing an additional avidin-biotin block should prevent staining of the exposed biotin when a biotin-based detection system is used.
Q. Can one do Antigen Retrieval and wait the next day to stain?
A. If one does not have time to complete the stain after Antigen Retrieval, the slides may be put in PBS overnight in the cold. PBS is slightly alkaline, and may cause some tissue to begin to fall off the slide. Care must be taken for any ER/PR studies, since there may be leaching. Comparative studies are always recommended.
Q. What if tissue is not fixed in formaldehyde? Is Antigen Retrieval needed?
A. Antigen Retrieval may also be needed with non cross-linking fixatives such as alcohol.
Q. If tissue is fixed in Bouin's or Gluteraldehyde, is Antigen Retrieval done the same way?
A. One can use the same general methods for Antigen Retrieval even if the tissue is fixed in Bouin's or Gluteraldehyde, but the times may have to be modified from the recommended range of 7 to 10 minutes. Bouin's is paraformaldehyde in unbuffered solution and probably would require less time. Gluteraldehyde is a super cross linker, and would probably need a longer period of time.
Q. Does one need Antigen Retrieval if the tissue is fixed in B5?
A. Antigen Retrieval is not needed for T and B cell markers, if the tissue is fixed in B5.
Q. Can Antigen Retrieval be done on over decalcified tissues?
A. The retrieval solution DeCal may be used to recover antigenicity in formalin-fixed, paraffin or celloidin-embedded, acid or EDTA-decalcified bone marrow. It is an immersion solution only and not for microwave use.
Q. Is the pH of the solution important?
A. There are many notations in the literature where pH is of great importance for the retrieval of antigens. Antibodies may react differently, some more optimally, when using antigen retrieval solutions with a different pH.
Q. How do I determine which Antigen Retrieval solution to use with my antibody?
A. BioGenex will inform the customer which Antigen Retrieval solution is recommended for any of its primary antibodies. When working with an antibody from another manufacturer, BioGenex offers the Retrievit™ Sampler Kit (Cat. # BK-1000-25). This kit provides the customer with five different Antigen Retrieval solutions so the customer can find out which one is the best for that particular antibody.
Q. Is Antigen Retrieval only necessary when performing IHC?
A. No. Antigen Retrieval is often also needed when doing ISH with DNA probes in addition to the treatment with Proteinase K. Antigen Retrieval is not recommended for ISH with mRNA probes.
