Many of you who have done immunohistochemistry (IHC) staining are familiar with antigen retrieval. This seemingly simple step has a significant impact on IHC experiments and often determines the success of IHC assays. Bioss summarized a series of tips for you as a scientific expert, from retrieval buffers, boiling devices, retrieval methods, and matters needing attention. We hope these small details can improve your IHC experiences and extend your scientific research life to be successful.
Antigen retrieval is an essential step for formaldehyde-fixed paraffin-embedded (FFPE) sections. Since the fixation process creates methylene bridges between amino acids leading to cross-linking of proteins, antigens (epitopes) of interest might be masked, resulting in poor recognition (interaction) of primary antibodies. To solve this problem, methods used to restore antigenicity and enhance antibody-epitope binding are called antigen retrial, and two commonly used methods are heat-induced antigen (epitope) retrieval (HIER) and Proteolytic Induced Epitope Retrieval (PIER).
Heat-Induced Epitope Retrieval (HIER)
There are a variety of buffers suitable for HIER, and we recommend two commonly used buffers: 10 mM citrate buffer (pH 6.0) and 0.5 mM EDTA buffer (pH 8.0).
Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0):
Tri-sodium citrate (dihydrate) --------- 2.94 g
Distilled water ------------------------ 1000 ml
Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 °C for longer storage.
Note: This buffer is commonly used and works perfectly with many antibodies. It gives very nice intense staining with a very low background.
EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0):
EDTA (CAS# 6381-92-6) ---------------- 0.37 g
Distilled water -------------------------- 1000 ml
Mix to dissolve. Adjust pH to 8.0 using 1N NaOH. Then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for three months or at 4 °C for longer storage.
Note: This buffer works excellent for many antibodies, but it often gives high background staining (maybe due to endogenous biotin revealed after this pretreatment). So primary antibodies can often be highly diluted. It is beneficial for low-affinity antibodies or when tissue antigens are not intense.
Note: This method is highly recommended. Three minutes is only a suggested time. You can set up a control experiment, set 1, 2, 3, 4... minutes to explore and determine the best recovery time for your antigens of interest. In addition, please make sure that the slides are completely cooled before proceeding to the next step.
Note: This method is convenient with drawbacks. In addition to the difficulties of regulating temperature, the longer retrieval time (20 minutes) may cause the sample dissociation from the slide. It is also crucial to ensure that the antigen retrieval buffer is sufficient to cover the slides (a few centimeters higher) to prevent drying out during “boiling”.
Note: The primary drawback of water baths is the inability to achieve retrieval temperatures above 100 °C, resulting in more extended protocols and several retrieval buffer evaporations during the process. Therefore, it is critical to ensure that the antigen retrieval buffer is sufficient to cover the slides (a few centimeters higher) to prevent drying out during “boiling”.
Proteolytic Induced Epitope Retrieval (PIER)
Trypsin is widely used for FFPE tissue sections, thus enhancing the staining intensity of antibodies.
Trypsin Working Solution (0.05%):
Trypsin ---------------------------------- 5 mg
Calcium chloride -----------------------10 mg
Distilled Water --------------------------10 ml
Mix to dissolve. Adjust pH to 7.8 with 1N N NaOH. Store at 4 ºC for one month or -20 ºC for long term storage
Note: Place slides on a plastic rack in the humidifier chamber to avoid touching directly the metal rack of the incubator. Otherwise, the staining quality will be affected by uneven temperature.