By Pamela A. Marcum, HT (ASCP), MS

The most commonly used fixative in histology today is 10% neutral buffered formalin (NBF). It has been used for the past 50 years and is the only fixative most laboratories will consider for routine tissue fixation. NBF is also the most common cause of protein cross-linking or denaturing during fixation of tissue. This cross-linking of proteins often blocks access to the antigenic sites required for immunohistochemistry (IHC) reactions, or staining. Since IHC is now the gold standard for diagnosis in cancer therapy, histologists must find a way to unmask antigens to achieve optimal IHC staining.

The most common methods for unmasking antigenic sites are enzymatic reactions with protease K, trypsin, or heat-induced epitope retrieval (HIER) with citrate buffer at various pH levels. The pH levels are determined by the antigen or epitope to be exposed. These methods are often very hard on the tissue and can cause the sections to be lifted off the slide, preventing further testing of the tissue section. A new chemical reaction method for exposing antigens and saving the tissue sections was developed to address these problems. This new method requires little or no heat during exposure and reduces the time required to complete IHC protocols by as much as 45 minutes. Using this new procedure, histotechnologists can get slides into the pathologist’s hands faster for quicker diagnosis and better patient prognosis.

The time savings is an obvious cost savings for the laboratory. The product is inexpensive and can be reused several times if the method is done in bulk versus the automated method where only 100 to 200 µL per slide is used.

 Difference in staining intensity between outer tissue layers and inner tissue layers due to improper fixation in 10% NBF. The outer tissue layers appear on the left side of the photo and are stained darker than the internal tissue layers on the right side of the photo. The sample is tonsil stained with CD30. Sample magnification 20X.

Conventional Methods of Unmasking Antigens
The first methods utilizing heat to unmask antigens in formalin-fixed paraffin embedded (FFPE) tissue samples were noted in the early 1990s by doctors Shan-Rong Shi, Mark Key, and Kris Kalra.1 This patented method, referred to as antigen retrieval, is performed in a microwave oven. The second most common method, heat induced epitope retrieval (HIER), is performed in a steamer or water bath. Prior to completing the IHC staining procedure, the histologist uses one of these methods to heat the tissue sections in a citrate buffer solution prepared at the required pH.

The histologist must preheat the citrate buffer to 95°–100°C prior to adding the slides, which takes 10 to 15 minutes. The deparaffinized slides then require 15 to 20 minutes to incubate and then another 20 to 30 minutes to cool. Most laboratories add the slides to the steamer when it reaches the correct temperature, and turn it down or off to avoid boiling the sections. With this method, the sections are sometimes damaged during the long exposure to heat and subsequent cool down. In addition, the procedure takes up to an hour to complete.

 IHC staining of estrogen receptor (Dako) after 5-minute incubation at room temperature with LAB Solution. Sample magnification 40X.  IHC staining of estrogen receptor (Dako) after a 20-minute incubation in citrate buffer at 100°C followed by a 20-minute cool down. Sample magnification 40X.
 IHC staining of progesterone receptor (Dako) after a 5-minute incubation at room temperature with LAB Solution. Sample magnification 40X.  IHC staining of progesterone receptor (Dako) after a 20-minute incubation in citrate buffer at 100°C followed by a 20-minute cool down. Sample magnification 40X.

Enzymatic methods of unmasking are completed with solutions containing either protease K, trypsin, or another enzyme in a specific strength to reduce the cross-linkage and expose the antigen. These solutions are most often purchased from companies specializing in IHC reagents and antibodies. They are sold at specific enzyme activity levels to avoid destroying the tissue. Protocols for making the solutions are available; however, it is critical to know the activity of the enzyme to assure the proper reaction.

A Superior Method
Polysciences Inc of Warrington, Pa, recently developed a new way to unmask antigens that can be completed faster and with less damage to the tissue sections. This method utilizes classical protein chemistry as a base and the liberating antibody binding (LAB) solution. Using this method, most samples are incubated at room temperature—only the most difficult antigens require heating to 60°C in a standard oven. This can save up to 40 minutes in staining time for many IHC reactions. In addition, tissue samples are exposed to the unmasking solution for a much shorter time, causing less section damage.

LAB Solution requires no additional equipment and can be used with either automated stainers or manual methods. Automated stainers require an additional step to be programmed for the application of the LAB Solution for 5 to 20 minutes, followed by a rinse. This is followed by the standard IHC protocols. Manual staining has an additional incubation step with LAB Solution, a rinse, and the standard protocol. When heating is required to expose difficult antigens, the incubation time will be 5 to 20 minutes at 60°C with no cool-down time required.

LAB Solution can be used with standard paraffin-embedded tissues, including melanoma and breast cancer panels, HPV, EBV, other cancers and many CD markers. Some very difficult antigens may require the heating step prior to completing the procedure.

Fixation Considerations
One of the major causes of poor staining is uneven fixation. This occurs when the external circumference is well fixed, but the center areas are poorly penetrated and fixed. Poor fixation may result from specimens grossed thicker than 3–4 mm. With thick specimens, the fixative may be unable to be fully penetrated during a routine processing program. Uneven fixation can cause very irregular or patchy final staining for both IHC reactions and routine stains, even when using unmasking protocols. Like other unmasking methods, the LAB Solution method also requires that specimens be well fixed for optimal results.

The sections in the photo at right show uneven staining due to poor fixative penetration. The peripheral edges are well stained and show positive cells, while deeper into the marginally fixed area the cells become less apparent, or negative.

Increasing the fixation times can result in more complete fixation, but also more cross-linking of proteins, which can cause poor IHC staining. This will not affect routine staining such as hematoxylin and eosin Y (H&E). There are two reasons that it is difficult to gauge fixative penetration: the penetration rate changes during fixation of the outer layers, and different types of tissue have different penetration rates. This problem can be avoided by grossing of initial specimen to a uniform size between 1-mm and no more than 4-mm thick. A rough standard of penetration for each tissue or organ system can evolve to help maintain the best possible fixation and staining. Table 1 provides a guideline of penetration rates and times for various types of tissues. Using tables of this type reduces the guesswork in determining the time required for fixation and sectioning.

Type of tissue Penetration rate Total time for 4 mm2
all routine surgical tissues 1.0mm/hour 5 to 6 hours
fatty, compact tissues
(breast, uterus)
0.8mm/hour 8 hours to overnight
These penetration rates are based on well-grossed specimens.

In summary, LAB Solution is an effective method for routine unmasking of antigens for IHC staining. This new method allows antigen unmasking in less time and at room temperature or much lower temperatures than other methods. Because of this, LAB Solution can be used on automated stainers whether they have heating capabilities or not. This new method is simpler and faster to use, cutting the time to get slides into the pathologist’s hand for quicker diagnosis and better patient prognosis.

Pamela A. Marcum, HT (ASCP), MS, is the histology/microscopy product development manager for Polysciences Inc.

1. Shi S, Key M, Kalra K. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6):741-748.