Abstract
Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris–EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method.
References
Bankfalvi A, Navabi H, Bier B, Bocker W, Jasani B, Schmid KW (1994) Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry. J Pathol 174(3):223–228. doi:10.1002/path.1711740312
Emoto K, Yamashita S, Okada Y (2005) Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens? J Histochem Cytochem 53(11):1311–1321. doi:10.1369/jhc.5C6627.2005
Hausen P, Dreyer C (1982) Urea reactivates antigens in paraffin sections for immunofluorescent staining. Stain Technol 57(5):321–324
Huang SN (1975) Immunohistochemical demonstration of hepatitis B core and surface antigens in paraffin sections. Lab Invest 33(1):88–95
Huang SN, Minassian H, More JD (1976) Application of immunofluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest 35(4):383–390
Kawai K, Serizawa A, Hamana T, Tsutsumi Y (1994) Heat-induced antigen retrieval of proliferating cell nuclear antigen and p53 protein in formalin-fixed, paraffin-embedded sections. Pathol Int 44(10–11):759–764
Kim SH, Kook MC, Shin YK, Park SH, Song HG (2004) Evaluation of antigen retrieval buffer systems. J Mol Histol 35(4):409–416
Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57(2):230–236
Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval. J Pathol 174(4):301–307. doi:10.1002/path.1711740410
Morgan JM, Navabi H, Jasani B (1997) Role of calcium chelation in high-temperature antigen retrieval at different pH values. J Pathol 182(2):233–237. doi:10.1002/(sici)1096-9896(199706)182:2<233:aid-path827>3.0.co;2-y
Norton AJ, Jordan S, Yeomans P (1994) Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 173(4):371–379. doi:10.1002/path.1711730413
Ramos-Vara JA, Beissenherz ME (2000) Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest 12(4):307–311
Roussel AJ, Knol AC, Bourdeau PJ, Bruet V (2014) Optimization of an immunohistochemical method to assess distribution of tight junction proteins in canine epidermis and adnexae. J Comp Pathol 150(1):35–46. doi:10.1016/j.jcpa.2013.06.008
Scalia CR, Gendusa R, Cattoretti G (2015) A 2-step Laemmli and antigen retrieval method improves immunodetection. Appl Immunohistochem Mol Morphol. doi:10.1097/pai.0000000000000203
Shi SR, Key ME, Kalra KL (1991) 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 39(6):741–748
Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43(2):193–201
Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49(8):931–937
Shi S, Cheng Q, Zhang P, Wang N, Zheng Y, Bai XY, Chen X (2013) Immunofluorescence with dual microwave retrieval of paraffin-embedded sections in the assessment of human renal biopsy specimens. Am J Clin Pathol 139(1):71–78. doi:10.1309/AJCPRZG8EXN7BAID
Song XF, Ren H, Andreasen A, Thomsen JS, Zhai XY (2012) Expression of Bcl-2 and Bax in mouse renal tubules during kidney development. PLoS One 7(2):e32771. doi:10.1371/journal.pone.0032771
Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem 71(5):263–270
Zhai XY, Kristoffersen IB, Christensen EI (2007) Immunocytochemistry of renal membrane proteins on epoxy sections. Kidney Int 72(6):731–735. doi:10.1038/sj.ki.5002403
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The work was financially supported by the National Natural Science Foundation of China (Contract No. 31371219).
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Gu, L., Cong, J., Zhang, J. et al. A microwave antigen retrieval method using two heating steps for enhanced immunostaining on aldehyde-fixed paraffin-embedded tissue sections. Histochem Cell Biol 145, 675–680 (2016). https://doi.org/10.1007/s00418-016-1426-7
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DOI: https://doi.org/10.1007/s00418-016-1426-7