TECHNICAL REPORT
Antigen Unmasking Induced by Superheating Marin Basic, MSc, Maja Jovicic Milentijevic, PhD, MD, David Basic, BSc, and Hakija Basic, PhD, MD
Abstract: This paper is dedicated to the description of superheating as a method for antigen retrieval. In our investigation, this antigen retrieval method was used on thermal plate, heating tissue sections at temperature 1201 C for 90 minutes. In the research we conducted the superheating method was applied to formalin-fixed paraffin-embedded tissue sections of breast tumors. The following monoclonal antibodies were used: estrogen receptor, progesterone receptor, epithelial membrane antigen, CD34, and Ki-67. With these tested antibodies we had good staining and no loss of tissue sections during the staining process. Key Words: immunohistochemistry, superheating, antigen retrieval, thermal plate (Appl Immunohistochem Mol Morphol 2014;22:774–776)
T
he major advances in immunohistochemistry (IHC) and their application in diagnostic pathology have occurred after the publication by Taylor and Burns,1 which reported that immunoperoxidase labeling can show the presence of
plasma cell immunoglobulin in paraffin-embedded tissues. It was shown that protoeolytic enzymes can strikingly enhance the intensity of staining of routinely processed paraffin-embedded tissue.2,3 The introduction of the antigen retrieval (AR) technique for the demonstration of antigens represented a new era in pathology laboratories.4 Following the development of the AR technique in 1991, hundreds of articles have been published worldwide documenting its application in IHC for the archival of formalin-fixed, paraffin-embedded tissue sections.5 The basic principle of different heat-induced AR methods is the fact that the intensity (temperature) and duration (time) of heating may critically influence the outcome.6 The technique of high temperature AR heating has since been adopted universally as a simple, effective, and reliable for routine IHC in surgical pathology.7
MATERIALS AND METHODS The study was based on patients diagnosed with invasive breast carcinoma. Sections of formalin-fixed, paraffin-embedded breast carcinoma were stained by us-
FIGURE 1. Estrogen receptor-positive breast carcinoma with and without antigen retrieval (alkaline phosphatase-anti alkaline phosphatase, 20 0.40).
Received for publication September 14, 2013; accepted October 20, 2013. From the Faculty of Medicine, University of Nis, Nis, Serbia. The authors declare no conflict of interest. Reprints: Maja Jovicic Milentijevic, PhD, MD, Faculty of Medicine, University of Nis, Tome Rosandica 3/11, 18000 Nis, Serbia (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
774 | www.appliedimmunohist.com
Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
Antigen Unmasking Induced by Superheating
FIGURE 2. Dermis (infiltrated by breast carcinoma) contains CD34 positive small vessels and control (alkaline phosphatase-anti alkaline phosphatase, 10 0.25).
ing immunoalkaline technique.8 IHC was performed by using the following monoclonal antibodies (DAKO): estrogen receptor (clone ER 1D5), progesterone receptor (clone PgR 636), epithelial membrane antigen (EMA), CD34, and Ki-67 (clone MIB-1). The sections in which AR was not performed served as a control. Paraffin sections were cut and attached to the not precoated glass slides and were air dried overnight at room temperature. AR was performed afterwards. Heat-induced AR was done by thermal plate at 1201 C (the thermometer was controlled at the slide surface) for 90 minutes. The paraffin melted in a few seconds. The tissue sections were placed in metal slide holder and the holder was put on a thermal plate, heated to 1201 C for 90 minutes. After AR, the slides were deparaffinized for 10 minutes in xylene, 10 minutes in acetone, and hydrated 10 minutes in dilution (1:1) of acetone and Tris-buffered saline. Finally, it was put in Tris-buffered saline until it was ready to stain. Then the slides were sequentially treated with diluted primary antibodies which had been incubated in the fridge overnight. The secondary antibody was incubated for 1 hour at room temperature. Alkaline phosphatase-anti alkaline phosphatase complex was applied for 1 hour. The new fuchsine was used as a chromogen, counterstaining with Giemsa.
formalin fixation was demonstrated by Shi et al.4 Higher temperature in general yields better results of AR-IHC. The optimal result of AR-IHC is correlated with the product of heating temperature (T ) time of AR heating treatment (t).9 So far different heating methods have been used, such as: water bath, microwaving, autoclaving, pressure cooking, and steam heating.5 Temperature of 1201 C, referred to as superheating AR, is frequently attained during heat-induced AR by using microwave, pressure cooker, or steaming methods.10 In the preliminary study we tested AR for 30, 60, and 90 minutes. EMA positivity was present in AR after 30 minutes; however, Ki-67 was positive after 60 minutes. Positive reaction for ER, PR, and CD34 was gained after 90-minute AR. In this study, we used thermal plate as a method to attain time and temperature for AR at 1201C. Microwave heating of histologic sections in citrate buffer is a widely used method of AR, but often results in a loss of tissue sections.11 With our method, we had good antigen recovery as well. There was no loss of tissue sections. Advantages of our method: quite simple and instrument inexpensive, no need for AR solution, no tissue sections loss. Disadvantage: long time AR.
RESULTS
REFERENCES
The levels of immunoreaction were graded into 3 groups: low, moderate, and strong expression. The stain intensity was significantly stronger with EMA. The sections stained with: ER, PR, CD34, and Ki-67, had moderate to strong staining. The proportion of tissue lost from each slide after staining was also assessed (Figs. 1, 2). It should be stressed that during the AR superheating, there is no loss of tissue sections.
1. Taylor CR, Burns J. Immunohistological detection of intracellular immunoglobulin containing cells in formalin fixed, paraffinembedded tissues using peroxidase labelled antibody. J Clin Pathol. 1974;27:14–20. 2. Huang SN, Minassian H, More JD. Application of immunofluorescent staining of paraffin sections improved by trypsin digestion. Lab Invest. 1976;35:383–390. 3. Reading M. A digestion technique for the reduction of background staining in the immunoperoxidase method. J Clin Pathol. 1977;30:80–90. 4. Shi SR, Key ME, Kalra KL. AR in formalin-fixed, paraffinembedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39:741–748. 5. Shi SR, Taylor CR. Antigen Retrieval Immunohistochemistry Based Research and Diagnostics. Hoboken, NJ: Wiley; 2010.
DISCUSSION The fact that high temperature heating is the most important factor for the retrieval of antigens masked by r
2014 Lippincott Williams & Wilkins
www.appliedimmunohist.com |
775
Basic et al
Appl Immunohistochem Mol Morphol
6. Taylor CR, Shi SR, Cote RJ. Antigen retrieval for immunohistochemistry status and need for greater standardization. Appl Immunohistochem. 1996;4:144–166. 7. Taylor CR, Cote RJ. Immunomicroscopy. A Diagnostic Tool for Surgical Pathologist. 3rd ed. Philadelphia: Elsevier Saunders; 2006. 8. Cordell J, Falini B, Erber W, et al. Immunoenzymatic label of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem. 1984;32:219–229.
776 | www.appliedimmunohist.com
Volume 22, Number 10, November/December 2014
9. Shi SR, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem. 1997;45:327–343. 10. Leong AS, Lee ES, Yin H, et al. Superheating antigen retrieval. Appl Immunohistochem Mol Morphol. 2002;10:263–268. 11. Frost AR, Sparks DBS, Grizzle WE. Methods of antigen recovery in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohistochem Mol Morphol. 2000;8: 236–243.
r
2014 Lippincott Williams & Wilkins
Antigen Unmasking Induced by Superheating Marin Basic, MSc, Maja Jovicic Milentijevic, PhD, MD, David Basic, BSc, and Hakija Basic, PhD, MD
Abstract: This paper is dedicated to the description of superheating as a method for antigen retrieval. In our investigation, this antigen retrieval method was used on thermal plate, heating tissue sections at temperature 1201 C for 90 minutes. In the research we conducted the superheating method was applied to formalin-fixed paraffin-embedded tissue sections of breast tumors. The following monoclonal antibodies were used: estrogen receptor, progesterone receptor, epithelial membrane antigen, CD34, and Ki-67. With these tested antibodies we had good staining and no loss of tissue sections during the staining process. Key Words: immunohistochemistry, superheating, antigen retrieval, thermal plate (Appl Immunohistochem Mol Morphol 2014;22:774–776)
T
he major advances in immunohistochemistry (IHC) and their application in diagnostic pathology have occurred after the publication by Taylor and Burns,1 which reported that immunoperoxidase labeling can show the presence of
plasma cell immunoglobulin in paraffin-embedded tissues. It was shown that protoeolytic enzymes can strikingly enhance the intensity of staining of routinely processed paraffin-embedded tissue.2,3 The introduction of the antigen retrieval (AR) technique for the demonstration of antigens represented a new era in pathology laboratories.4 Following the development of the AR technique in 1991, hundreds of articles have been published worldwide documenting its application in IHC for the archival of formalin-fixed, paraffin-embedded tissue sections.5 The basic principle of different heat-induced AR methods is the fact that the intensity (temperature) and duration (time) of heating may critically influence the outcome.6 The technique of high temperature AR heating has since been adopted universally as a simple, effective, and reliable for routine IHC in surgical pathology.7
MATERIALS AND METHODS The study was based on patients diagnosed with invasive breast carcinoma. Sections of formalin-fixed, paraffin-embedded breast carcinoma were stained by us-
FIGURE 1. Estrogen receptor-positive breast carcinoma with and without antigen retrieval (alkaline phosphatase-anti alkaline phosphatase, 20 0.40).
Received for publication September 14, 2013; accepted October 20, 2013. From the Faculty of Medicine, University of Nis, Nis, Serbia. The authors declare no conflict of interest. Reprints: Maja Jovicic Milentijevic, PhD, MD, Faculty of Medicine, University of Nis, Tome Rosandica 3/11, 18000 Nis, Serbia (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins
774 | www.appliedimmunohist.com
Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
Antigen Unmasking Induced by Superheating
FIGURE 2. Dermis (infiltrated by breast carcinoma) contains CD34 positive small vessels and control (alkaline phosphatase-anti alkaline phosphatase, 10 0.25).
ing immunoalkaline technique.8 IHC was performed by using the following monoclonal antibodies (DAKO): estrogen receptor (clone ER 1D5), progesterone receptor (clone PgR 636), epithelial membrane antigen (EMA), CD34, and Ki-67 (clone MIB-1). The sections in which AR was not performed served as a control. Paraffin sections were cut and attached to the not precoated glass slides and were air dried overnight at room temperature. AR was performed afterwards. Heat-induced AR was done by thermal plate at 1201 C (the thermometer was controlled at the slide surface) for 90 minutes. The paraffin melted in a few seconds. The tissue sections were placed in metal slide holder and the holder was put on a thermal plate, heated to 1201 C for 90 minutes. After AR, the slides were deparaffinized for 10 minutes in xylene, 10 minutes in acetone, and hydrated 10 minutes in dilution (1:1) of acetone and Tris-buffered saline. Finally, it was put in Tris-buffered saline until it was ready to stain. Then the slides were sequentially treated with diluted primary antibodies which had been incubated in the fridge overnight. The secondary antibody was incubated for 1 hour at room temperature. Alkaline phosphatase-anti alkaline phosphatase complex was applied for 1 hour. The new fuchsine was used as a chromogen, counterstaining with Giemsa.
formalin fixation was demonstrated by Shi et al.4 Higher temperature in general yields better results of AR-IHC. The optimal result of AR-IHC is correlated with the product of heating temperature (T ) time of AR heating treatment (t).9 So far different heating methods have been used, such as: water bath, microwaving, autoclaving, pressure cooking, and steam heating.5 Temperature of 1201 C, referred to as superheating AR, is frequently attained during heat-induced AR by using microwave, pressure cooker, or steaming methods.10 In the preliminary study we tested AR for 30, 60, and 90 minutes. EMA positivity was present in AR after 30 minutes; however, Ki-67 was positive after 60 minutes. Positive reaction for ER, PR, and CD34 was gained after 90-minute AR. In this study, we used thermal plate as a method to attain time and temperature for AR at 1201C. Microwave heating of histologic sections in citrate buffer is a widely used method of AR, but often results in a loss of tissue sections.11 With our method, we had good antigen recovery as well. There was no loss of tissue sections. Advantages of our method: quite simple and instrument inexpensive, no need for AR solution, no tissue sections loss. Disadvantage: long time AR.
RESULTS
REFERENCES
The levels of immunoreaction were graded into 3 groups: low, moderate, and strong expression. The stain intensity was significantly stronger with EMA. The sections stained with: ER, PR, CD34, and Ki-67, had moderate to strong staining. The proportion of tissue lost from each slide after staining was also assessed (Figs. 1, 2). It should be stressed that during the AR superheating, there is no loss of tissue sections.
1. Taylor CR, Burns J. Immunohistological detection of intracellular immunoglobulin containing cells in formalin fixed, paraffinembedded tissues using peroxidase labelled antibody. J Clin Pathol. 1974;27:14–20. 2. Huang SN, Minassian H, More JD. Application of immunofluorescent staining of paraffin sections improved by trypsin digestion. Lab Invest. 1976;35:383–390. 3. Reading M. A digestion technique for the reduction of background staining in the immunoperoxidase method. J Clin Pathol. 1977;30:80–90. 4. Shi SR, Key ME, Kalra KL. AR in formalin-fixed, paraffinembedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39:741–748. 5. Shi SR, Taylor CR. Antigen Retrieval Immunohistochemistry Based Research and Diagnostics. Hoboken, NJ: Wiley; 2010.
DISCUSSION The fact that high temperature heating is the most important factor for the retrieval of antigens masked by r
2014 Lippincott Williams & Wilkins
www.appliedimmunohist.com |
775
Basic et al
Appl Immunohistochem Mol Morphol
6. Taylor CR, Shi SR, Cote RJ. Antigen retrieval for immunohistochemistry status and need for greater standardization. Appl Immunohistochem. 1996;4:144–166. 7. Taylor CR, Cote RJ. Immunomicroscopy. A Diagnostic Tool for Surgical Pathologist. 3rd ed. Philadelphia: Elsevier Saunders; 2006. 8. Cordell J, Falini B, Erber W, et al. Immunoenzymatic label of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem. 1984;32:219–229.
776 | www.appliedimmunohist.com
Volume 22, Number 10, November/December 2014
9. Shi SR, Cote RJ, Taylor CR. Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem. 1997;45:327–343. 10. Leong AS, Lee ES, Yin H, et al. Superheating antigen retrieval. Appl Immunohistochem Mol Morphol. 2002;10:263–268. 11. Frost AR, Sparks DBS, Grizzle WE. Methods of antigen recovery in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohistochem Mol Morphol. 2000;8: 236–243.
r
2014 Lippincott Williams & Wilkins
