https://doi.org/10.4081/ejh.2024.3929
Walking a thin line between fixation and epitope binding – characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue
Submitted: 29 November 2023
Accepted: 19 March 2024
Published: 16 April 2024
Accepted: 19 March 2024
Abstract Views: 581
PDF: 254
HTML: 7
HTML: 7
Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
Antibody-based fluorescence analysis of female reproductive tissues in research of sexually transmitted diseases allows for an in-depth understanding of protein localization, interactions, and pathogenesis. However, in many cases, cryosectioning is not compatible with biosafety regulations; at all times, exposure of lab personnel and the public to potentially harmful pathogens from biological infectious material must be avoided; thus, formaldehyde fixation is essential. Due to formaldehyde’s cross-linking properties, protein detection with antibodies can be impeded. To allow effective epitope binding during immunofluorescence of formalin-fixed paraffin-embedded vaginal tissue, we investigated two antigen retrieval methods. We tested these methods regarding their suitability for automated image analysis, facilitating reproducible quantitative microscopic data acquisition in sexually transmitted disease research. Heat-based retrieval at 80°C in citrate buffer proved to increase antibody binding to eosinophil protein and HSV-2 visibly and tissue morphology best, and was the most efficient for sample processing and quantitative analysis.
Fox CH, Johnson FB, Whiting J, Roller PP. Formaldehyde fixation. J Histochem Cytochem 1985;33:845-53.
DOI: https://doi.org/10.1177/33.8.3894502
Musiał A, Gryglewski RW, Kielczewski S, Loukas M, Wajda J. Formalin use in anatomical and histological science in the 19th and 20th centuries. Folia Med Cracov 2016;56:31-40.
Hoetelmans RWM, Prins FA, Velde IC, van der Meer J, van de Velde CJH, van Dierendonck JH. Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. Appl Immunohist Mol Morphol 2001;9:346-51.
DOI: https://doi.org/10.1097/00129039-200112000-00010
Möller L, Schünadel L, Nitsche A, Schwebke I, Hanisch M, Laue M. Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy. Viruses 2015;7:666-79.
DOI: https://doi.org/10.3390/v7020666
Battifora H. Quality assurance issues in immunohistochemistry. J Histotechnol 1999;22:16-75.
DOI: https://doi.org/10.1179/his.1999.22.3.169
Hoetelmans RW, van Slooten HJ, Keijzer R, van de Velde CJ, van Dierendonck JH. Routine formaldehyde fixation irreversibly reduces immunoreactivity of Bcl-2 in the nuclear compartment of breast cancer cells, but not in the cytoplasm. Appl Immunohist Mol Morphol 2001;9:74-80.
DOI: https://doi.org/10.1097/00022744-200103000-00014
Miettinen M. Immunostaining of intermediate filament proteins in paraffin sections. Pathol Res Pract 1989;184:431-6.
DOI: https://doi.org/10.1016/S0344-0338(89)80039-1
Shi SR, Key ME, Kalra KL. 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;39:741-8.
DOI: https://doi.org/10.1177/39.6.1709656
Seidal T, Balaton AJ, Battifora H. Interpretation and quantification of immunostains. Am J Surg Pathol 2001;25:1204.
DOI: https://doi.org/10.1097/00000478-200109000-00013
Im K, Mareninov S, Diaz MFP, Yong WH. An introduction to performing immunofluorescence staining. Methods Mol Biol 2019;1897:299-311.
DOI: https://doi.org/10.1007/978-1-4939-8935-5_26
De Cock H, Ducatelle R, Logghe JP. Immunohistochemical localisation of estrogen receptor in the normal canine female genital tract. Domest Anim Endocrinol 1997;14:133-47.
DOI: https://doi.org/10.1016/S0739-7240(97)00001-5
Pudney J, Quayle AJ, Anderson DJ. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone1. Biol Reprod 2005;73:1253-63.
DOI: https://doi.org/10.1095/biolreprod.105.043133
Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem 1993;41:1599-604.
DOI: https://doi.org/10.1177/41.11.7691930
Al-Qaoud KM, Taubert A, Zahner H, Fleischer B, Hoerauf A. Infection of BALB/c mice with the filarial nematode Litomosoides sigmodontis: role of CD4+ T cells in controlling larval development. Infect Immun 1997;65:2457-61.
DOI: https://doi.org/10.1128/iai.65.6.2457-2461.1997
Volkmann L, Bain O, Saeftel M, Specht S, Fischer K, Brombacher F, et al. Murine filariasis: interleukin 4 and interleukin 5 lead to containment of different worm developmental stages. Med Microbiol Immunol 2003;192:23-31.
DOI: https://doi.org/10.1007/s00430-002-0155-9
Chetty A, Darby MG, Vornewald PM, Martín-Alonso M, Filz A, Ritter M et al. Il4ra-independent vaginal eosinophil accumulation following helminth infection exacerbates epithelial ulcerative pathology of HSV-2 infection. Cell Host Microbe 2021;29:579-93.e5.
DOI: https://doi.org/10.1016/j.chom.2021.02.004
Nada R, Kumar A, Kumar VG, Gupta KL, Joshi K. Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies. Indian J Nephrol 2016;26:182-7.
DOI: https://doi.org/10.4103/0971-4065.159558
Krenacs L, Krenacs T, Stelkovics E, Raffeld M. Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections. Methods Mol Biol 2010;588:103-19.
DOI: https://doi.org/10.1007/978-1-59745-324-0_14
Shi SR, Liu C, Taylor CR. Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis. J Histochem Cytochem 2006;55:105-9.
DOI: https://doi.org/10.1369/jhc.6P7080.2006
Frost AR, Sparks D, Grizzle WE. Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohist Mol Morphol 2000;8:236-43.
DOI: https://doi.org/10.1097/00129039-200009000-00011
Hecke DV. Routine Immunohistochemical staining today: choices to make, challenges to take. J Histotechnol 2002;25:45-54.
DOI: https://doi.org/10.1179/his.2002.25.1.45
D’Amico F, Skarmoutsou E, Stivala F. State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 2009;341:1-18.
DOI: https://doi.org/10.1016/j.jim.2008.11.007
Ethics Approval
All protocols and experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, GermanySupporting Agencies
German Research Foundation (DFG) within the German-African Projects in Infectiology, German Center for Infection Research (DZIF), Bundesministerium fur Bildung und ForschungHow to Cite
Wadephul, L. M., Arndts, K., Katawa, G., Dietlmeier, E., Horsnell, W., Hoerauf, A., & Ritter, M. (2024). Walking a thin line between fixation and epitope binding – characterization of antigen retrieval methods suitable for eosinophil and HSV-2 staining in formalin-fixed female reproductive tissue. European Journal of Histochemistry, 68(2). https://doi.org/10.4081/ejh.2024.3929
Copyright (c) 2024 The Author(s)
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
PAGEPress has chosen to apply the Creative Commons Attribution NonCommercial 4.0 International License (CC BY-NC 4.0) to all manuscripts to be published.
Similar Articles
- D. Martini, A. Trirè, L. Breschi, A. Mazzoni, G. Teti, M. Falconi, A. Ruggeri Jr, Dentin matrix protein 1 and dentin sialophosphoprotein in human sound and carious teeth: an immunohistochemical and colorimetric assay , European Journal of Histochemistry: Vol. 57 No. 4 (2013)
- M. Costanzo, F. Carton, A. Marengo, G. Berlier, B. Stella, S. Arpicco, M. Malatesta, Fluorescence and electron microscopy to visualize the intracellular fate of nanoparticles for drug delivery , European Journal of Histochemistry: Vol. 60 No. 2 (2016)
- Simona Polakovičová, Mária Csӧbӧnyeiová, Barbora Filova, Miroslav Borovský, Ladislav Maršík, Alena Kvasilová, Štefan Polák, Merkel-like cell distribution in the epithelium of the human vagina. An immunohistochemical and TEM study , European Journal of Histochemistry: Vol. 62 No. 1 (2018)
- Yao Le, Zhijun Wang, Qian Zhang, Ling Miao, Xiaohong Wang, Guorong Han, Study on the mechanism of Shenling Baizhu powder on the pathogenesis of pregnancy complicated with non-alcoholic fatty liver, based on PI3K/AKT/mTOR signal pathway , European Journal of Histochemistry: Vol. 68 No. 3 (2024)
- K.R. Percival, Z. A. Radi, A modified Verhoeff-van Gieson elastin histochemical stain to enable pulmonary arterial hypertension model characterization , European Journal of Histochemistry: Vol. 60 No. 1 (2016)
- J. Ning, J. Zhang, W. Liu, Y. Lang, Y. Xue, S. Xu, Overexpression of ubiquitin-specific protease 22 predicts poor survival in patients with early-stage non-small cell lung cancer , European Journal of Histochemistry: Vol. 56 No. 4 (2012)
- M. Malatesta, C. Zancanaro, M. Costanzo, B. Cisterna, C. Pellicciari, Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabeling in cultured cells , European Journal of Histochemistry: Vol. 57 No. 3 (2013)
- Y. Zhang, W.Y. Ye, J.Q. Wang, S.J. Wang, P. Ji, G.Y. Zhou, G.P. Zhao, H.L. Ge, Y. Wang, dCTP pyrophosphohydrase exhibits nucleic accumulation in multiple carcinomas , European Journal of Histochemistry: Vol. 57 No. 3 (2013)
- Y. Zhang, Y.J. Tang, Z.H. Li, F. Pan, K. Huang, G.H. Xu, KiSS1 inhibits growth and invasion of osteosarcoma cells through inhibition of the MAPK pathway , European Journal of Histochemistry: Vol. 57 No. 4 (2013)
- Kazuhiko Hashimoto, Shunji Nishimura, Tomohiko Ito, Masao Akagi, Characterization of PD-1/PD-L1 immune checkpoint expression in soft tissue sarcomas , European Journal of Histochemistry: Vol. 65 No. 3 (2021)
<< < 1 2 3 4 5 6 7 8 9 10 > >>
You may also start an advanced similarity search for this article.
