Современные технологии фиксации биологического материала, применяемые при проведении иммуногистохимических исследований (обзор)

На основе собственного опыта и данных литературы рассмотрены современные способы фиксации биологического материала, используемые при проведении иммуногистохимических исследований. Среди необходимых свойств иммуногистохимического фиксатора на первом месте должны быть способность обеспечивать сохранность структуры ткани при наименьшем влиянии на антигенные свойства макромолекул. С этой точки зрения в обзоре анализируется применимость для иммуногистохимии ряда фиксаторов, широко используемых в гистологической, цитологической и иммуногистохимической практике: альдегидов (формальдегид, глутаральдегид, глиоксаль), обезвоживающих (коагулирующих) фиксаторов (этанол, метанол, ацетон), комбинированных фиксирующих растворов (жидкость Буэна, жидкость Карнуа, метакарн и др.), а также современных цинксодержащих фиксаторов и коммерческих продуктов, предлагаемых для фиксации биологических образцов. Использование большинства фиксаторов ведет к нарушению третичной и четвертичной структуры многих белков, что требует для их выявления с помощью иммуногистохимии дополнительной процедуры демаскирования эпитопов с помощью протеолитических ферментов или повышенной температуры. На основании анализа данных иммуногистохимических исследований различных антигенов отмечено высокое качество нового цинксодержащего фиксатора — цинк-этанол-формальдегида. Тем не менее сделан вывод, что ни один из известных на сегодняшний день фиксаторов не обладает таким сочетанием свойств, которые бы позволяли получать гистологические препараты высокого качества и при этом обеспечивали возможность выявления любых антигенов в изучаемой ткани.

1. Коржевский Д.Э. Фиксация материала для гистоло­ги­ческого исследования. В кн.: Морфологическая диагнос­тика: подготовка материала для морфологического ис­сле­дования и электронной микроскопии. Под ред. Кор­жевского Д.Э. СПб; 2013; c. 10–25.
2. Kiernan J.A. Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Micros Today 2000; 8(01): 8–13, https://doi.org/10.1017/s1551929500057060.
3. Walker J.F. Formaldehyde. New York: Reinhold; 1964.
4. Dapson R.W. Macromolecular changes caused by formalin fixation and antigen retrieval. Biotech Histochem 2007; 82(3): 133–140, https://doi.org/10.1080/10520290701567916.
5. Helander K.G. Kinetic studies of formaldehyde binding in tissue. Biotech Histochem 1994; 69(3): 177–179, https://doi.org/10.3109/10520299409106282.
6. Jamur M.C., Oliver C. Cell fixatives for immunostaining. Methods Mol Biol 2010; 588: 55–61, https://doi.org/10.1007/978-1-59745-324-0_8.
7. Leong A.S., Gilham P.N. The effects of progressive formaldehyde fixation on the preservation of tissue antigens. Pathology 1989; 21(4): 266–268, https://doi.org/10.3109/00313028909061071.
8. Masuda N., Ohnishi T., Kawamoto S., Monden M., Okubo K. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res 1999; 27(22): 4436–4443, https://doi.org/10.1093/nar/27.22.4436.
9. Puchtler H., Meloan S.N. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 1985; 82(3): 201–204, https://doi.org/10.1007/bf00501395.
10. Griffiths G. Fine structure immunocytochemistry. Springer Berlin Heidelberg; 1993, https://doi.org/10.1007/978-3-642-77095-1.
11. Hopwood D. Theoretical and practical aspects of glutaraldehyde fixation. In: Fixation in histochemistry. Stoward P.J. (editor). Springer US; 1973; p. 47–83, https://doi.org/10.1007/978-1-4899-3260-0_2.
12. Southern L.J., Hughes H., Lawford P.V., Clench M.R., Manning N.J. Glutaraldehyde-induced cross-links: a study of model compounds and commercial bioprosthetic valves. J Heart Valve Dis 2000; 9(2): 241–249.
13. Jang W., Choi S., Kim S.H., Yoon E., Lim H.G., Kim Y.J. A comparative study on mechanical and biochemical properties of bovine pericardium after single or double crosslinking treatment. Korean Circ J 2012; 42(3): 154–163, https://doi.org/10.4070/kcj.2012.42.3.154.
14. Werkmeister J.A., Tebb T.A., Peters D.E., Ramshaw J.A. The use of quenching agents to enable immunofluorescent examination of collagen-based biomaterials showing glutaraldehyde-derived autofluorescence. Clinical Materials 1990; 6(1): 13–20, https://doi.org/10.1016/0267-6605(90)90040-3.
15. Callis G. Glutaraldehyde-induced autofluorescence. Biotech Histochem 2010; 85(4): 269–270, https://doi.org/10.3109/10520290903472415.
16. Hayat M.A. Principles and techniques of electron microscopy: biological applications. Cambridge: Cambridge University Press, 2000.
17. Kiernan J.A. Histological and histochemical methods: theory and practice. Oxford: Butterworth-Heinemann; 1999.
18. Lee K., Choi S., Yang C., Wu H.C., Yu J. Autofluorescence generation and elimination: a lesson from glutaraldehyde. Chem Commun (Camb) 2013; 49(29): 3028–3030, https://doi.org/10.1039/c3cc40799c.
19. Ruzin S.E. Plant microtechnique and microscopy. Oxford, New York: Oxford University Press; 1999.
20. Schelkle K.M., Schmid C., Yserentant K., Bender M., Wacker I., Petzoldt M., Hamburger M., Herten D.P., Wombacher R., Schröder R.R., Bunz U.H. Cell fixation by light-triggered release of glutaraldehyde. Angew Chem Int Ed Engl 2017; 56(17): 4724–4728, https://doi.org/10.1002/ange.201612112.
21. Constantinou P., Dacosta R.S., Wilson B.C. Extending immunofluorescence detection limits in whole paraffin-embedded formalin fixed tissues using hyperspectral confocal fluorescence imaging. J Microsc 2009; 234(2): 137–146, https://doi.org/10.1111/j.1365-2818.2009.03155.x.
22. Del Castillo P., Llorente A.R., Stockert J.C. Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis. Basic Appl Histochem 1989; 33(3): 251–257.
23. Kajimura J., Ito R., Manley N.R., Hale L.P. Optimization of single- and dual-color immunofluorescence protocols for formalin-fixed, paraffin-embedded archival tissues. J Histochem Cytochem 2016; 64(2): 112–124, https://doi.org/10.1369/0022155415610792.
24. Robertson D., Isacke C.M. Multiple immunofluorescence labeling of formalin-fixed paraffin-embedded tissue. Methods Mol Biol 2011; 724: 69–77, https://doi.org/10.1007/978-1-61779-055-3_4.
25. Suetterlin R., Baschong W., Laeng R.H. Immunofluorescence and confocal laser scanning microscopy of chronic myeloproliferative disorders on archival formaldehyde-fixed bone marrow. J Histochem Cytochem 2004; 52(3): 347–354, https://doi.org/10.1177/002215540405200305.
26. Galkina М.V., Snopova L.B., Prodanets N.N., Lapshin R.D., Belousova I.I., Abrosimov D.A., Bugrova М.L. Atrial and brain natriuretic peptides of secretory cardiomyocytes in salt loading in experiment. Sovremennye tehnologii v medicine 2016; 8(3): 49–55, https://doi.org/10.17691/stm2016.8.3.05.
27. Grigor’ev I.P. Ultrastructural reorganizations in the rat cerebral cortex after injection of ascorbic acid into the ventricular fluid. Neurosci Behav Physiol 1989; 19(6): 529–534, https://doi.org/10.1007/bf01181871.
28. Mrini A., Moukhles H., Jacomy H., Bosler O., Doucet G. Efficient immunodetection of various protein antigens in glutaraldehyde-fixed brain tissue. J Histochem Cytochem 1995; 43(12): 1285–1291, https://doi.org/10.1177/43.12.8537644.
29. Отеллин В.А., Неокесарийский А.А., Коржевский Д.Э. Изменения структуры ядра нейронов некортекса при де­фи­ците серотонина и катехоламинов. Цитология 1998; 40(4): 256–259.
30. Bilinski S.M., Jaglarz M.K., Dougherty M.T., Kloc M. Electron microscopy, immunostaining, cytoskeleton visualization, in situ hybridization, and three-dimensional reconstruction of Xenopus oocytes. Methods 2010; 51(1): 11–19, https://doi.org/10.1016/j.ymeth.2009.12.003.
31. De Paul A.L., Mukdsi J.H., Petiti J.P., Gutiérrez S., Quintar A.A., Maldonado C.A., Torres A.I. Immunoelectron microscopy: a reliable tool for the analysis of cellular processes. Applications of Immunocytochemistry 2012, https://doi.org/10.5772/33108.
32. Winey M., Meehl J.B., O’Toole E.T., Giddings T.H. Jr. Conventional transmission electron microscopy. Mol Biol Cell 2014; 25(3): 319–323, https://doi.org/10.1091/mbc.e12-12-0863.
33. Baum H.P., Reichrath J., Theobald A., Schock G. Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections. Histochem J 1994; 26(12): 929–933, https://doi.org/10.1007/bf00174008.
34. D’Amico F., Skarmoutsou E., Stivala F. State of the art in antigen retrieval for immunohistochemistry. J Immunol Methods 2009; 341 (1–2): 1–18, https://doi.org/10.1016/j.jim.2008.11.007.
35. Eltoum I., Fredenburgh J., Grizzle W.E. Advanced concepts in fixation: 1. Effects of fixation on immunohistochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues. 2. Developmental methods of fixation. J Histotechnol 2001; 24(3): 201–210, https://doi.org/10.1179/his.2001.24.3.201.
36. Mason J.T., O’Leary T.J. Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem 1991; 39(2): 225–229, https://doi.org/10.1177/39.2.1987266.
37. Meyer W., Hornickel N. Tissue fixation — the most underestimated methodical feature of immunohistochemistry. In: Méndez-Vilas A., Díaz J. (editors). Microscopy: science, technology, applications and education. Vol. 2. Formatex. Microscopy Series. Badajoz: Formatex Research Center; 2010; p. 953–959.
38. Polak J.M., van Noorden S. Introduction to immunocytochemistry. Oxford: BIOS Scientific Publishers; 2003.
39. Shi S.R., Shi Y., Taylor C.R. Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades. J Histochem Cytochem 2011; 59(1): 13–32, https://doi.org/10.1369/jhc.2010.957191.
40. Vincek V., Nassiri M., Nadji M., Morales A.R. A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples. Lab Invest 2003; 83(10): 1427–1435, https://doi.org/10.1097/01.lab.0000090154.55436.d1.
41. Wine Y., Cohen-Hadar N., Freeman A., Frolow F. Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis. Biotechnol Bioeng 2007; 98(3): 711–718, https://doi.org/10.1002/bit.21459.
42. De Marzo A.M., Fedor H.H., Gage W.R., Rubin M.A. Inadequate formalin fixation decreases reliability of p27 immunohistochemical staining: probing optimal fixation time using high-density tissue microarrays. Hum Pathol 2002; 33(7): 756–760, https://doi.org/10.1053/hupa.2002.126187.
43. Fritschy J.M. Is my antibody-staining specific? How to deal with pitfalls of immunohistochemistry. Eur J Neurosci 2008; 28(12): 2365–2370, https://doi.org/10.1111/j.1460-9568.2008.06552.x.
44. Oyama T., Ishikawa Y., Hayashi M., Arihiro K., Horiguchi J. The effects of fixation, processing and evaluation criteria on immunohistochemical detection of hormone receptors in breast cancer. Breast Cancer 2007; 14 (2): 182–188, https://doi.org/10.2325/jbcs.976.
45. Ramos-Vara J.A. Technical aspects of immunohistochemistry. Vet Pathol 2005; 42(4): 405–426, https://doi.org/10.1354/vp.42-4-405.
46. Watanabe J., Asaka Y., Kanamura S. Relationship between immunostaining intensity and antigen content in sections. J Histochem Cytochem 1996; 44(12): 1451–1458, https://doi.org/10.1177/44.12.8985137.
47. Grizzle W.E., Stockard C.R., Billings P.E. The effects of tissue processing variables other than fixation on histochemical staining and immunohistochemical detection of antigens. J Histotechnol 2001; 24(3): 213–219, https://doi.org/10.1179/his.2001.24.3.213.
48. Hayat M.A. Microscopy, immunohistochemistry, and antigen retrieval methods: for light and electron microscopy. Springer US; 2002, https://doi.org/10.1007/b112626.
49. Otali D., Stockard C.R., Oelschlager D.K., Wan W., Manne U., Watts S.A., Grizzle W.E. Combined effects of formalin fixation and tissue processing on immunorecognition. Biotech Histochem 2009; 84(5): 223–247, https://doi.org/10.3109/10520290903039094.
50. Muñoz de Toro de Luque M., Luque E.H. Effect of microwave pretreatment on proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections. J Histotechnol 1995; 18(1): 11–16, https://doi.org/10.1179/his.1995.18.1.11.
51. Коржевский Д.Э., Гиляров А.В. Протеолитическое и тепловое демаскирование антигенов. В кн.: Теоретические основы и практическое применение методов иммуно­гисто­химии. Под ред. Д.Э. Коржевского. СПб; 2012; c. 30–35.
52. Коржевский Д.Э., Юмкина Е.А. Применение метода теплового демаскирования антигенов на парафиновых сре­зах головного мозга крысы. Морфология 2005; 127(2): 76–77.
53. Leong T.Y., Leong A.S. How does antigen retrieval work? Adv Anat Pathol 2007; 14(2): 129–131, https://doi.org/10.1097/pap.0b013e31803250c7.
54. Boenisch T. Heat-induced antigen retrieval: what are we retrieving? J Histochem Cytochem 2006; 54(9): 961–964, https://doi.org/10.1369/jhc.6p6945.2006.
55. Shi S.-R., Key M.E., Kalra K.L. Antigen retrieval in formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991; 39(6): 741–748, https://doi.org/10.1177/39.6.1709656.
56. Shi S.-R., Cote R.J., Taylor C.R. Antigen retrieval techniques: current perspectives. J Histochem Cytochem 2001; 49(8): 931–937, https://doi.org/10.1177/002215540104900801.
57. Gill S.K., Ishak M., Rylett R.J. Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: detection of NeuN. J Neurosci Methods 2005; 148(1): 26–35, https://doi.org/10.1016/j.jneumeth.2005.03.008.
58. Dapson R.W. Glyoxal fixation: how it works and why it only occasionally needs antigen retrieval. Biotech Histochem 2007; 82(3): 161–166, https://doi.org/10.1080/10520290701488113.
59. Dapson R.W., Feldman A.T., Wolfe D. Glyoxal fixation and its relationship to immunohistochemistry. J Histotechnol 2006; 29(2): 65–76, https://doi.org/10.1080/01478885.2006.11800879.
60. Arnold M.M., Srivastava S., Fredenburgh J., Stockard C.R., Myers R.B., Grizzle W.E. Effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens. Biotech Histochem 1996; 71(5): 224–230, https://doi.org/10.3109/10520299609117164.
61. Suzuki M., Katsuyama K., Adachi K., Ogawa Y., Yorozu K., Fujii E., Misawa Y., Sugimoto T. Combination of fixation using PLP fixative and embedding in paraffin by the AMeX method is useful for histochemical studies in assessment of immunotoxicity. J Toxicol Sci 2002; 27(3): 165–172, https://doi.org/10.2131/jts.27.165.
62. Otali D., He Q., Stockard C.R., Grizzle W.E. Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol. Biotech Histochem 2013; 88(3–4): 170–180, https://doi.org/10.3109/10520295.2012.754496.
63. Otali D., He Q., Grizzle W.E. The effect of antigen retrieval on cells fixed in 10% neutral buffered formalin followed by transfer to 70% ethanol. PLoS One 2013; 8(12): e82405, https://doi.org/10.1371/journal.pone.0082405.
64. Hoetelmans R.W., Prins F.A., Cornelese-ten Velde I., van der Meer J., van de Velde C.J., van Dierendonck J.H. Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. Appl Immunohistochem Mol Morphol 2001; 9(4): 346–351, https://doi.org/10.1097/00129039-200112000-00010.
65. Korzhevskii D.E., Lentsman M.V., Kirik O.V., Otellin V.A. Vimentin-immunopositive cells in the rat telencephalon after experimental ischemic stroke. Neurosci Behav Physiol 2008; 38(8): 845–848, https://doi.org/10.1007/s11055-008-9061-y.
66. Giaccone G., Canciani B., Puoti G., Rossi G., Goffredo D., Iussich S., Fociani P., Tagliavini F., Bugiani O. Creutzfeldt-Jakob disease: Carnoy’s fixative improves the immunohistochemistry of the proteinase K-resistant prion protein. Brain Pathol 2000; 10(1): 31–37, https://doi.org/10.1111/j.1750-3639.2000.tb00240.x.
67. Pereira M.A., Dias A.R., Faraj S.F., Cirqueira Cdos S., Tomitao M.T., Nahas S.C., Ribeiro U. Jr., de Mello E.S. Carnoy’s solution is an adequate tissue fixative for routine surgical pathology, preserving cell morphology and molecular integrity. Histopathology 2015; 66(3): 388–397, https://doi.org/10.1111/his.12532.
68. Shetye J.D., Scheynius A., Mellstedt H.T., Biberfeld P. Retrieval of leukocyte antigens in paraffin-embedded rat tissues. J Histochem Cytochem 1996; 44(7): 767–776, https://doi.org/10.1177/44.7.8675998.
69. Yoneyama M., Kitayama T., Taniura H., Yoneda Y. Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus. J Neurosci Res 2003; 73(3): 416–426, https://doi.org/10.1002/jnr.10622.
70. James J.D., Hauer-Jensen M. Effects of fixative and fixation time for quantitative computerized image analysis of immunohistochemical staining. J Histotechnol 1999; 22(2): 109–111, https://doi.org/10.1179/his.1999.22.2.109.
71. Bos P.K., van Osch G.J., van der Kwast T., Verwoerd-Verhoef H.L., Verhaar J.A. Fixation-dependent immunolocalization shift and immunoreactivity of intracellular growth factors in cartilage. Histochem J 2000; 32(7): 391–396, https://doi.org/10.1023/a:1004023902080.
72. Shi S.R., Liu C., Pootrakul L., Tang L., Young A., Chen R., Cote R.J., Taylor C.R. Evaluation of the value of frozen tissue section used as “gold standard” for immunohistochemistry. Am J Clin Pathol 2008; 129(3): 358–366, https://doi.org/10.1309/7cxuyxt23e5al8kq.
73. Sillevis Smitt P.A., van der Loos C., Vianney de Jong J.M, Troost D. Tissue fixation methods alter the immunohistochemical demonstrability of neurofilament proteins, synaptophysin, and glial fibrillary acidic protein in human cerebellum. Acta Histochem 1993; 95(1): 13–21, https://doi.org/10.1016/s0065-1281(11)80381-8.
74. Eastwood S.L., Burnet P.W., McDonald B., Clinton J., Harrison P.J. Synaptophysin gene expression in human brain: a quantitative in situ hybridization and immunocytochemical study. Neuroscience 1994; 59(4): 881–892, https://doi.org/10.1016/0306-4522(94)90292-5.
75. Webster J.D., Miller M.A., Dusold D., Ramos-Vara J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem 2009; 57(8): 753–761, https://doi.org/10.1369/jhc.2009.953877.
76. Wehrl H.F., Bezrukov I., Wiehr S., Lehnhoff M., Fuchs K., Mannheim J.G., Quintanilla-Martinez L., Kohlhofer U., Kneilling M., Pichler B.J., Sauter A.W. Assessment of murine brain tissue shrinkage caused by different histological fixatives using magnetic resonance and computed tomography imaging. Histol Histopathol 2015; 30(5): 601–613.
77. Schmidt J., Bodor O., Gohr L., Kunz W. Paramyosin isoforms of Schistosoma mansoni are phosphorylated and localized in a large variety of muscle types. Parasitology 1996; 112(5): 459–467, https://doi.org/10.1017/s0031182000076927.
78. Beckstead J.H. A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues. J Histochem Cytochem 1994; 42(8): 1127–1134, https://doi.org/10.1177/42.8.8027531.
79. Ismail J.A., Poppa V., Kemper L.E., Scatena M., Giachelli C.M., Coffin J.D., Murry C.E. Immunohistologic labeling of murine endothelium. Cardiovasc Pathol 2003; 12(2): 82–90, https://doi.org/10.1016/s1054-8807(02)00166-7.
80. Lynn J.A., Whitaker B.P., Hladik C.L., Robinson R.J., Joie J.B., Stigliano W.W., Carson F.L. Zinc isopropyl alcoholic unbuffered formalin as a postfixative for routine surgical pathology specimens. J Histotechnol 1994; 17(2): 105–109, https://doi.org/10.1179/014788894794710986.
81. Ott S.R. Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts. J Neurosci Methods 2008; 172(2): 220–230, https://doi.org/10.1016/j.jneumeth.2008.04.031.
82. Wester K., Asplund A., Bäckvall H., Micke P., Derveniece A., Hartmane I., Malmström P.U., Pontén F. Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues. Lab Invest 2003; 83(6): 889–899, https://doi.org/10.1097/01.lab.0000074892.53211.a5.
83. Accart N., Sergi F., Rooke R. Revisiting fixation and embedding techniques for optimal detection of dendritic cell subsets in tissues. J Histochem Cytochem 2014; 62(9): 661–671, https://doi.org/10.1369/0022155414539963.
84. Коржевский Д.Э., Григорьев И.П., Отеллин В.А. При­менение обезвоживающих фиксаторов, содержащих со­ли цинка, в нейрогистологических исследованиях. Морфо­ло­гия 2006; 129(1): 85–86.
85. Korzhevskii D.E., Sukhorukova E.G., Gilerovich E.G., Petrova E.S., Kirik O.V., Grigor’ev I.P. Advantages and disadvantages of zinc-ethanol-formaldehyde as a fixative for immunocytochemical studies and confocal laser microscopy. Neurosci Behav Physiol 2014; 44(5): 542–545, https://doi.org/10.1007/s11055-014-9948-8.
86. Korzhevskii D.E., Sukhorukova E.G., Kirik O.V., Grigorev I.P. Immunohistochemical demonstration of specific antigens in the human brain fixed in zinc-ethanol-formaldehyde. Eur J Histochem 2015; 59(3): 2530, https://doi.org/10.4081/ejh.2015.2530.
87. Alekseeva O.S., Vetosh A.N., Kostkin V.B., Korzhevskiy D.E., Otellin V.A. Heat shock proteins in brain neurons and hypoxic preconditioning. Dokl Biol Sci 2009; 425(1): 98–100, https://doi.org/10.1134/s0012496609020045.
88. Gilerovich E.G., Fedorova E.A., Grigor’ev I.P., Korzhevskii D.E. Morphological basics for reorganization of the rat cerebellar cortex during senescence. J Evol Biochem Physiol 2015; 51(5): 421–427, https://doi.org/10.1134/s0022093015050087.
89. Grigor’ev I.P., Korzhevskii D.E., Sukhorukova E.G., Gusel’nikova V.V., Kirik O.V. Intranuclear ubiquitin-immunopositive structures in human substantia nigra neurons. Cell and Tissue Biology 2016; 10(1): 29–36, https://doi.org/10.1134/s1990519x16010053.
90. Grigoriev I.P., Vasilenko M.S., Sukhorukova E.G., Korzhevskii D.E. Use of different antibodies to tyrosine hydroxylase to study catecholaminergic systems in the mammalian brain. Neurosci Behav Physiol 2012; 42(2): 210–213, https://doi.org/10.1007/s11055-011-9555-x.
91. Kirik O.V., Korzhevskii D.E. Expression of neural stem cell marker nestin in the kidney of rats and humans. Bull Exp Biol Med 2009; 147(4): 539–541, https://doi.org/10.1007/s10517-009-0541-z.
92. Kirik O.V., Korzhevskii D.E. Vimentin in ependymal and subventricular proliferative zone cells of rat telencephalon. Bull Exp Biol Med 2013; 154(4): 553–557, https://doi.org/10.1007/s10517-013-1998-3.
93. Kirik O.V., Grigorev I.P., Alekseeva O.S., Korzhevskii D.E. Three-dimensional organization of the cytoplasmic neuroglobin-immunopositive structures in the rat brainstem neurons. Biochem Moscow Suppl Ser A 2016; 10(4): 333–337, https://doi.org/10.1134/s1990747816030065.
94. Korzhevskii D.E., Grigor’ev I.P., Kirik O.V., Alekseeva O.S. Neuroglobin distribution in the rat cerebellar Purkinje cells. J Evol Biochem Phys 2015 51(6): 517–519, https://doi.org/10.1134/s0022093015060095.
95. Коржевский Д.Э., Григорьев И.П., Сухорукова Е.Г., Гусельникова В.В. Иммуногистохимическая характеристика нейронов черного вещества головного мозга человека. Журнал неврологии психиатрии им. С.С. Корсакова 2017; 117 (4): 50–55, https://doi.org/10.17116/jnevro20171174150-55.
96. Petrova E.S., Isaeva E.N., Korzhevskii D.E. Differentiation of dissociated rat embryonic brain after allotransplantation into damaged nerve. Bull Exp Biol Med 2013; 156(1): 136–138, https://doi.org/10.1007/s10517-013-2296-9.
97. Chumasov E.I., Korzhevskii D.E., Petrova E.S., Sapronov N.S., Kuznetsova N.N. Glial reaction of the subventricular zone of the telencephalon of the rat brain on modeling of Alzheimer’s disease. Neurosci Behav Physiol 2012; 42(1): 67–71, https://doi.org/10.1007/s11055-011-9535-1.
98. Chumasov E.I., Petrova E.S., Korzhevskii D.E. Distribution and structural organization of the autonomic nervous apparatus in the rat pancreas (an immunohistochemical study). Neurosci Behav Physiol 2012; 42(8): 781–788, https://doi.org/10.1007/s11055-012-9635-6.
99. Benerini Gatta L., Cadei M., Balzarini P., Castriciano S., Paroni R., Verzeletti A., Cortellini V., De Ferrari F., Grigolato P. Application of alternative fixatives to formalin in diagnostic pathology. Eur J Histochem 2012; 56(2): e12, https://doi.org/10.4081/ejh.2012.12.
100. Lassalle S., Hofman V., Marius I., Gavric-Tanga V., Brest P., Havet K., Butori C., Selva E., Santini J., Mograbi B., Hofman P. Assessment of morphology, antigenicity, and nucleic acid integrity for diagnostic thyroid pathology using formalin substitute fixatives. Thyroid 2009; 19(11): 1239–1248, https://doi.org/10.1089/thy.2009.0095.
101. Moelans C.B., ter Hoeve N., van Ginkel J.W., ten Kate F.J., van Diest P.J. Formaldehyde substitute fixatives. Analysis of macroscopy, morphologic analysis, and immunohistochemical analysis. Am J Clin Pathol 2011; 136(4): 548–556, https://doi.org/10.1309/ajcphh1b0cocbgom.
102. Nadji M., Nassiri M., Vincek V., Kanhoush R., Morales A.R. Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system. Appl Immunohistochem Mol Morphol 2005; 13(3): 277–282, https://doi.org/10.1097/01.pai.0000146544.51771.79.
103. Nykänen M., Kuopio T. Protein and gene expression of estrogen receptor alpha and nuclear morphology of two breast cancer cell lines after different fixation methods. Exp Mol Pathol 2010; 88(2): 265–271, https://doi.org/10.1016/j.yexmp.2009.12.003.
104. Olert J., Wiedorn K.H., Goldmann T., Kuhl H., Mehraein Y., Scherthan H., Niketeghad F., Vollmer E., Müller A.M., Müller-Navia J. HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues. Pathol Res Pract 2001; 197(12): 823–826, https://doi.org/10.1078/0344-0338-00166.
105. Paavilainen L., Edvinsson A., Asplund A., Hober S., Kampf C., Pontén F., Wester K. The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. J Histochem Cytochem 2010; 58(3): 237–246, https://doi.org/10.1369/jhc.2009.954321.
106. Preusser M., Plumer S., Dirnberger E., Hainfellner J.A., Mannhalter C. Fixation of brain tumor biopsy specimens with RCL2 results in well-preserved histomorphology, immunohistochemistry and nucleic acids. Brain Pathol 2010; 20(6): 1010–1020, https://doi.org/10.1111/j.1750-3639.2010.00400.x.
107. Robinson R.W., Snyder J.A. An innovative fixative for cytoskeletal components allows high resolution in colocalization studies using immunofluorescence techniques. Histochem Cell Biol 2004; 122(1): 1–5, https://doi.org/10.1007/s00418-004-0656-2.
108. Vollmer E., Galle J., Lang D.S., Loeschke S., Schultz H., Goldmann T. The HOPE technique opens up a multitude of new possibilities in pathology. Rom J Morphol Embryol 2006; 47(1): 15–19.
109. Zanini C., Gerbaudo E., Ercole E., Vendramin A., Forni M. Evaluation of two commercial and three home-made fixatives for the substitution of formalin: a formaldehyde-free laboratory is possible. Environ Health 2012; 11: 59, https://doi.org/10.1186/1476-069x-11-59.
110. Hinova-Palova D.V., Landzhov B., Dzhambazova E., Minkov M., Edelstein L., Malinova L., Paloff A., Ovtscharoff W. Neuropeptide Y immunoreactivity in the cat claustrum: a light- and electron-microscopic investigation. J Chem Neuroanat 2014; 61–62: 107–119, https://doi.org/10.1016/j.jchemneu.2014.08.007.
111. Persson S., Havton L.A. Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods. J Neurosci Methods 2009; 184(1): 42–47, https://doi.org/10.1016/j.jneumeth.2009.07.017.
112. Watanabe I.S., Dias F.J., Mardegan Issa J.P., dos Santos Haemmerle C.A., Cury D.P., Takada S.H., Sosthenes M.C., Pereira da Silva M.C., Campos L.M., Nogueira M.I., Iyomasa M.M. Immunohistochemistry and ultrastructural characteristics of nerve endings in the oral mucosa of rat. Microscopy 2013; 62(2): 259–270, https://doi.org/10.1093/jmicro/dfs068.
113. Stirling J.W. Ultrastructural localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution. J Histochem Cytochem 1989; 37(5): 709–714, https://doi.org/10.1177/37.5.2467929.
114. Koga D., Kusumi S., Bochimoto H., Watanabe T., Ushiki T. Correlative light and scanning electron microscopy for observing the three-dimensional ultrastructure of membranous cell organelles in relation to their molecular components. J Histochem Cytochem 2015; 63(12): 968–979, https://doi.org/10.1369/0022155415609099.
115. Berryman M.A. Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry. J Histochem Cytochem 1992; 40(6): 845–857, https://doi.org/10.1177/40.6.1350287.
116. Phend K.D., Rustioni A., Weinberg R.J. An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. J Histochem Cytochem 1995; 43(3): 283–292, https://doi.org/10.1177/43.3.7532656.
117. Zhong L., Brown J.C., Wells C., Gerges N.Z. Post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures. J Vis Exp 2013; 74: e50273, https://doi.org/10.3791/50273.
118. Heck W.L., Slusarczyk A., Basaraba A.M., Schweitzer L. Subcellular localization of GABA receptors in the central nervous system using post-embedding immunohistochemistry. Brain Res Brain Res Protoc 2002; 9(3): 173–180, https://doi.org/10.1016/s1385-299x(02)00143-5.
119. Kinugasa S., Tojo A., Sakai T., Fujita T. Silver-enhanced immunogold scanning electron microscopy using vibratome sections of rat kidneys: detection of albumin filtration and reabsorption. Med Mol Morphol 2010; 43(4): 218–225, https://doi.org/10.1007/s00795-010-0500-9.

Grigorev I.P., Korzhevskii D.E. Current Technologies for Fixation of Biological Material for Immunohistochemical Analysis (Review). Sovremennye tehnologii v medicine 2018; 10(2): 156, https://doi.org/10.17691/stm2018.10.2.19