Heat-Induced Epitope Retrieval (HIER)
The need to employ heat-induced epitope retrieval (HIER) as a routine procedure before the commencement of any immunostaining protocol cannot be overemphasized. Since the seminal work of Shi et al (1991), it has been recognized that the heating of deparaffinized tissue sections in a variety of retrieval solutions, up to boiling temperature, results in the `unmasking' of a very wide range of tissue antigens (Leong & Milios, 1993a, b; Gown et al, 1993, Catorretti et al, 1993; Cuevas et al, 1994). Tissues fixed in formaldehyde as well as a variety of common fixatives respond to this treatment with enhancement of a wide range of antigens (Byron, 1997), making it an indispensable requirement of immunolabeling. The method is equally applicable to cytological preparations and cell blocks(Suthipintawong et al, 1996, 1997).
Various solutions have been employed for antigen retrieval but a 10 mM solution of citrate buffer at pH 6.0 is the best universal reagent and can be prepared in the laboratory (2.1 g citric acid monohydrate perlitre of water, adjusted to pH 6.0 with NaOH) (Leong et al, 1996). Besides citrate buffer and other `home-made' retrieval solutions, several commercial reagents are available, including Antigen Retrieval Solution (Biogenex), Target Retrieval Solution (Dako), Target Unmasking Fluid (Monosan) and Target Unmasking Fluid (Serotec). We found that all the commercial reagents enhanced immunolabeling but no single reagent was consistently best for all diagnostic antibodies. The Dako product had a slight edge over other reagents including citrate buffer and EDTA at pH 8.0 (Leong et al, 1996). An important observation was that all reagents, including commercial retrieval solutions, were reusable for as many as 20 times without appreciable loss of reactivity, allowing considerable cost savings should commercial reagents be employed.
Shi et al (1995) suggested that pH is an important variable in epitope retrieval solutions and grouped antigens into three categories based on their reactivity at different pH. Antigens like CD 20, PCNA, AE1, EMA and NSE showed excellent retrieval throughout the pH range. MIB1 and estrogen receptor were immunoreactive following treatment in solutions of very low pH and at neutral to high pH, but displayed a dramatic drop of immunoreactivity at moderately acidic pH (pH 3-6). The third group, comprising antibodies such as MT1 (CD 43) and HMB-45, showed increasing intensities of staining in solutions of increasing pH and only weak staining at acidic pH.
Other variables such as temperature, duration of heating, composition of the retrieval solution, including molarity, salt content and presence of metallic ions, and the nature of the antigen of interest may influence immunoreactivity and should be considered when testing a new antibody. Heat is considered to be the major factor responsible for the `unmasking' of the epitopes `hidden' as a result of protein crosslinking induced by formaldehyde. Microwave irradiation was the first form of heating introduced by Shi et al (1991) as it produces instantaneous and uniform heating in small pieces of biological tissues (Leong et al, 1985). Other methods for generating heat have been employed for antigen retrieval including the conventional water bath, wet autoclaving, steaming, pressure cooking, hot oven heating in a humidified chamber (reviewed by Taylor et al, 1996) and even the sauna bath (A M Gown, personal communication). The most recent method of generating heat for antigen retrieval is the DNA thermal cycler (Baker-Cairns et al, 1996). While the virtues of each method have been argued, we find microwaves to be the most convenient.
Recently, antigen retrieval has been successfully achieved with ultrasound (Portiansky & Gimeno, 1996). As ultrasound generates negligible amounts of heat, this raises doubts about whether heat is a requirement of the procedure or if molecular kinetics is the mechanism of action. Another area of controversy lies in the concept of `unmasking' of antigenic epitopes produced by crosslinking fixatives. Interestingly, the demonstration that the antigen retrieval procedure also enhances immunoreactivity in tissues exposed to fixatives such as Zenker's and Carnoy's solutions (Byron, 1997), which are not considered to act by protein crosslinkage, raises more questions as to the mechanism of antigen retrieval.
Protocol for Heat-Induced Antigen Retrieval Using Microwaves
1.Mount 5 micron formalin-fixed, paraffin-embedded sections on aminoalkylsilane-coated slides.
2.Deparaffinize in xylene and rehydrate through graded alcohols.
3.Rinse in deionized water followed by rinsing in phosphate-buffered saline (PBS).
4.Block endogenous peroxidase with 0.5% hydrogen peroxide/methanol for 30 min.
5.Wash in PBS.
6.Stack slides on edge in a plastic container (Kartell, Milan, Italy). The transparent container allows stacking of up to 20 slides.
7.Fill the container with 10 mM citrate buffer at pH 6.0, taking care to completely immerse all tissue sections (the container takes about 250 ml of buffer).
8.Cover the container (the Kartell box is provided with a fitting lid that prevents boiling over).
9.Place the container with slides in a microwave oven that has a carousel (NEC model 702, 650 watts). If more than one container is used, place them equidistant at the periphery of the carousel.
10.Irradiate at maximum setting until boiling (about 5 min).
11.As soon as boiling is attained, adjust the power setting so that the solution simmers. Simmer for a further 10 min.
12.Turn off power. Allow the tissue sections to remain in the hot buffer for another 25 min.
13.Remove sections. Block in 3% non-immune horse serum for 20 min before incubating with the primary antibody. Immunostain with a standard immunoenzyme technique.
Note
The duration of heating depends on the fixation of the tissue sections. Over fixed tissues will require longer periods of heating, whereas inadequately fixed tissue sections will detach from the slides. Appropriate adjustments will be necessary to optimize this step. Immunoreactivity of some antigens can be further enhanced when HIER is performed in special commercial retrieval solutions (Leong et al, 1996) or by variation of the pH of the retrieval solution (Shi et al, 1995) and these will require evaluation especially when detecting antigens of low immunoreactivity.
Proteolytic digestion may enhance the immunoreactivity of some tissue antigens. The optimal stage at which to apply proteolytic enzymes will vary according to the antigen of interest; for example, with E-cadherin, proteolytic digestion is best performed before HIER, whereas for collagen type IV, laminin and cytokeratins, proteolytic digestion is optimally performed after HIER and just before incubation with the primary antibody. As with the duration of heating, the type, concentration of enzyme and duration of digestion will require optimization to suit the antigen of interest as well as the fixation of the tissue.
References
•Baker-Cairns B, Meyers K, Hamilton R et al 1996 Immunohistochemical staining of fixed tissue using antigen retrieval and a thermal cycler. Biotechniques 20: 641-650.
•Byron NA 1997 Antigen retrieval on paraffin-embedded tissue fixed with various fixatives. Journal of Cellular Pathology 2: 53-66.
•Catorretti C, Pilieri S, Parraviccini C et al 1993 Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. Journal of Pathology 171: 79-80.
•Cuevas EC, Bateman AC, Wilkins BS et al 1994 Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. Journal of Clinical Pathology 47:448-452.
•Gown AM, De Wever N, Battifora H 1993 Microwave-based antigen unmasking. A revolutionary new technique for routine immunohistochemistry. Applied Immunohistochemistry 1:256-266.
•Leong AS-Y, Milios J 1993a An assessment of the efficacy of the microwave-antigen retrieval procedure on a range of tissue antigens. Applied Immunohistochemistry 1:267-274.
•Leong AS-Y, Milios 1993b Comparison of antibodies to estrogen and progesterone receptors and the influence of microwave antigen retrieval. Applied Immunohistochemistry 1:282-288.
•Leong AS-Y, Dayman ME, Milios J 1985 Microwave irradiation as a form of fixation for light and electron microscopy. Journal of Pathology 146:313-321.
•Leong AS-Y, Milios J, Leong FJW-M 1996 Epitope retrieval with microwaves. A comparison of citrate buffer and EDTA with three commercial retrieval solutions. Applied Immunohistochemistry 4:201-207.
•Portiansky EL, Gimeno EJ 1996 A new epitope retrieval method for the detection of structural cytokeratins in the bovine prostatic tissue. Applied Immunohistochemistry 4:208-214.
•Shi S-R, Key ME, Kalra KL 1991 Antigen retireval in formalin-fixed, paraffin-embedded tissues. An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Journal of Histochemistry and Cytochemistry 39:741-748.
•Shi S-R, Iman A, Young L et al 1995 Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. Journal of Histochemistry and Cytochemistry 43: 193-201.
•Suthipintawong C, Leong AS-Y, Vinyuvat S 1996 Immunostaining of cell preparations. A comparative evaluation of common fixatives and protocols. Diagnostic Cytopathology 15: 167-174.
•Suthipintawong C, Leong AS-Y, Chan K-W, Vinyuvat S 1997 Immunostaining of estrogen receptor, progesterone receptor, MIB1 antigen, and c-erbB-2 oncoprotein in cytologic specimens: a simplified method with formalin fixation. Diagnostic Cytopathology 17: 127-133.
•Taylor CR, Shi S-R, Cote RJ 1996 Antigen retrieval for immunohistochemistry. Status and need for greater standardization. Applied Immunohistochemistry 4: 144-166.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
The need to employ heat-induced epitope retrieval (HIER) as a routine procedure before the commencement of any immunostaining protocol cannot be overemphasized. Since the seminal work of Shi et al (1991), it has been recognized that the heating of deparaffinized tissue sections in a variety of retrieval solutions, up to boiling temperature, results in the `unmasking' of a very wide range of tissue antigens (Leong & Milios, 1993a, b; Gown et al, 1993, Catorretti et al, 1993; Cuevas et al, 1994). Tissues fixed in formaldehyde as well as a variety of common fixatives respond to this treatment with enhancement of a wide range of antigens (Byron, 1997), making it an indispensable requirement of immunolabeling. The method is equally applicable to cytological preparations and cell blocks(Suthipintawong et al, 1996, 1997).
Various solutions have been employed for antigen retrieval but a 10 mM solution of citrate buffer at pH 6.0 is the best universal reagent and can be prepared in the laboratory (2.1 g citric acid monohydrate perlitre of water, adjusted to pH 6.0 with NaOH) (Leong et al, 1996). Besides citrate buffer and other `home-made' retrieval solutions, several commercial reagents are available, including Antigen Retrieval Solution (Biogenex), Target Retrieval Solution (Dako), Target Unmasking Fluid (Monosan) and Target Unmasking Fluid (Serotec). We found that all the commercial reagents enhanced immunolabeling but no single reagent was consistently best for all diagnostic antibodies. The Dako product had a slight edge over other reagents including citrate buffer and EDTA at pH 8.0 (Leong et al, 1996). An important observation was that all reagents, including commercial retrieval solutions, were reusable for as many as 20 times without appreciable loss of reactivity, allowing considerable cost savings should commercial reagents be employed.
Shi et al (1995) suggested that pH is an important variable in epitope retrieval solutions and grouped antigens into three categories based on their reactivity at different pH. Antigens like CD 20, PCNA, AE1, EMA and NSE showed excellent retrieval throughout the pH range. MIB1 and estrogen receptor were immunoreactive following treatment in solutions of very low pH and at neutral to high pH, but displayed a dramatic drop of immunoreactivity at moderately acidic pH (pH 3-6). The third group, comprising antibodies such as MT1 (CD 43) and HMB-45, showed increasing intensities of staining in solutions of increasing pH and only weak staining at acidic pH.
Other variables such as temperature, duration of heating, composition of the retrieval solution, including molarity, salt content and presence of metallic ions, and the nature of the antigen of interest may influence immunoreactivity and should be considered when testing a new antibody. Heat is considered to be the major factor responsible for the `unmasking' of the epitopes `hidden' as a result of protein crosslinking induced by formaldehyde. Microwave irradiation was the first form of heating introduced by Shi et al (1991) as it produces instantaneous and uniform heating in small pieces of biological tissues (Leong et al, 1985). Other methods for generating heat have been employed for antigen retrieval including the conventional water bath, wet autoclaving, steaming, pressure cooking, hot oven heating in a humidified chamber (reviewed by Taylor et al, 1996) and even the sauna bath (A M Gown, personal communication). The most recent method of generating heat for antigen retrieval is the DNA thermal cycler (Baker-Cairns et al, 1996). While the virtues of each method have been argued, we find microwaves to be the most convenient.
Recently, antigen retrieval has been successfully achieved with ultrasound (Portiansky & Gimeno, 1996). As ultrasound generates negligible amounts of heat, this raises doubts about whether heat is a requirement of the procedure or if molecular kinetics is the mechanism of action. Another area of controversy lies in the concept of `unmasking' of antigenic epitopes produced by crosslinking fixatives. Interestingly, the demonstration that the antigen retrieval procedure also enhances immunoreactivity in tissues exposed to fixatives such as Zenker's and Carnoy's solutions (Byron, 1997), which are not considered to act by protein crosslinkage, raises more questions as to the mechanism of antigen retrieval.
Protocol for Heat-Induced Antigen Retrieval Using Microwaves
1.Mount 5 micron formalin-fixed, paraffin-embedded sections on aminoalkylsilane-coated slides.
2.Deparaffinize in xylene and rehydrate through graded alcohols.
3.Rinse in deionized water followed by rinsing in phosphate-buffered saline (PBS).
4.Block endogenous peroxidase with 0.5% hydrogen peroxide/methanol for 30 min.
5.Wash in PBS.
6.Stack slides on edge in a plastic container (Kartell, Milan, Italy). The transparent container allows stacking of up to 20 slides.
7.Fill the container with 10 mM citrate buffer at pH 6.0, taking care to completely immerse all tissue sections (the container takes about 250 ml of buffer).
8.Cover the container (the Kartell box is provided with a fitting lid that prevents boiling over).
9.Place the container with slides in a microwave oven that has a carousel (NEC model 702, 650 watts). If more than one container is used, place them equidistant at the periphery of the carousel.
10.Irradiate at maximum setting until boiling (about 5 min).
11.As soon as boiling is attained, adjust the power setting so that the solution simmers. Simmer for a further 10 min.
12.Turn off power. Allow the tissue sections to remain in the hot buffer for another 25 min.
13.Remove sections. Block in 3% non-immune horse serum for 20 min before incubating with the primary antibody. Immunostain with a standard immunoenzyme technique.
Note
The duration of heating depends on the fixation of the tissue sections. Over fixed tissues will require longer periods of heating, whereas inadequately fixed tissue sections will detach from the slides. Appropriate adjustments will be necessary to optimize this step. Immunoreactivity of some antigens can be further enhanced when HIER is performed in special commercial retrieval solutions (Leong et al, 1996) or by variation of the pH of the retrieval solution (Shi et al, 1995) and these will require evaluation especially when detecting antigens of low immunoreactivity.
Proteolytic digestion may enhance the immunoreactivity of some tissue antigens. The optimal stage at which to apply proteolytic enzymes will vary according to the antigen of interest; for example, with E-cadherin, proteolytic digestion is best performed before HIER, whereas for collagen type IV, laminin and cytokeratins, proteolytic digestion is optimally performed after HIER and just before incubation with the primary antibody. As with the duration of heating, the type, concentration of enzyme and duration of digestion will require optimization to suit the antigen of interest as well as the fixation of the tissue.
References
•Baker-Cairns B, Meyers K, Hamilton R et al 1996 Immunohistochemical staining of fixed tissue using antigen retrieval and a thermal cycler. Biotechniques 20: 641-650.
•Byron NA 1997 Antigen retrieval on paraffin-embedded tissue fixed with various fixatives. Journal of Cellular Pathology 2: 53-66.
•Catorretti C, Pilieri S, Parraviccini C et al 1993 Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. Journal of Pathology 171: 79-80.
•Cuevas EC, Bateman AC, Wilkins BS et al 1994 Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies. Journal of Clinical Pathology 47:448-452.
•Gown AM, De Wever N, Battifora H 1993 Microwave-based antigen unmasking. A revolutionary new technique for routine immunohistochemistry. Applied Immunohistochemistry 1:256-266.
•Leong AS-Y, Milios J 1993a An assessment of the efficacy of the microwave-antigen retrieval procedure on a range of tissue antigens. Applied Immunohistochemistry 1:267-274.
•Leong AS-Y, Milios 1993b Comparison of antibodies to estrogen and progesterone receptors and the influence of microwave antigen retrieval. Applied Immunohistochemistry 1:282-288.
•Leong AS-Y, Dayman ME, Milios J 1985 Microwave irradiation as a form of fixation for light and electron microscopy. Journal of Pathology 146:313-321.
•Leong AS-Y, Milios J, Leong FJW-M 1996 Epitope retrieval with microwaves. A comparison of citrate buffer and EDTA with three commercial retrieval solutions. Applied Immunohistochemistry 4:201-207.
•Portiansky EL, Gimeno EJ 1996 A new epitope retrieval method for the detection of structural cytokeratins in the bovine prostatic tissue. Applied Immunohistochemistry 4:208-214.
•Shi S-R, Key ME, Kalra KL 1991 Antigen retireval in formalin-fixed, paraffin-embedded tissues. An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. Journal of Histochemistry and Cytochemistry 39:741-748.
•Shi S-R, Iman A, Young L et al 1995 Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. Journal of Histochemistry and Cytochemistry 43: 193-201.
•Suthipintawong C, Leong AS-Y, Vinyuvat S 1996 Immunostaining of cell preparations. A comparative evaluation of common fixatives and protocols. Diagnostic Cytopathology 15: 167-174.
•Suthipintawong C, Leong AS-Y, Chan K-W, Vinyuvat S 1997 Immunostaining of estrogen receptor, progesterone receptor, MIB1 antigen, and c-erbB-2 oncoprotein in cytologic specimens: a simplified method with formalin fixation. Diagnostic Cytopathology 17: 127-133.
•Taylor CR, Shi S-R, Cote RJ 1996 Antigen retrieval for immunohistochemistry. Status and need for greater standardization. Applied Immunohistochemistry 4: 144-166.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
