Comparative Examination of Commonly Used Some Fixatives with Routine Histochemical Staining’s for The Optimal Histological Appearance in The Gill Tissue of Zebrafish

Abstract

Histopathological studies related to Zebrafish which are used as a model organism in researching the pathogenesis of many diseases increase day by day. Because of the increasing importance of zebrafish research, experiments on this animal model are more prominent and gill tissue is frequently examined in various disease models using zebrafish. As it is known, at the end of the experimental animal model studies, if the histopathological examination is to be done, the tissues should be fixed. The purpose of fixation is to keep cellular and extracellular components in vivo as much as possible. Therefore, the choice of fixation methods and fixatives has a significant effect on tissue processing. In this study, we aimed to optimize fixation techniques and staining protocols for producing ideal slides and histological images of gill tissue in zebrafish. In our study, to determine the optimal histology in the zebrafish-gill tissue, the tissues were fixed with Bouin’s, Carnoy’s, 10% neutral buffered formalin (NBF), Davidson’s, and Dietrich’s solutions. Following the routine tissue processing, the sections were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome (MT) stains. Consequently, tissue morphology was best preserved with Bouin’s and NBF solutions. The best results in H&E stain were obtained from tissues fixed with NBF solution, while Dietrich's solution fixation was found to be ideal for MT staining.

Keywords

• Fixatives,Hematoxylin and Eosin,Masson’s Trichrome,Zebrafish,Gill

Zebra Balığı Solungaç Dokusunda Optimal Histolojik Görünüm için Rutin Histokimyasal Boyama ile Sık Kullanılan Fiksatiflerin Karşılaştırmalı Olarak İncelenmesi

Abstract

Birçok hastalığın patogenezinin araştırılmasında model bir organizma olarak kullanılan Zebra balığı ile ilgili histopatolojik çalışmalar gün geçtikçe artmaktadır. Zebra balığı araştırmalarının artan önemi nedeniyle, bu hayvan modelindeki deneyler daha önemlidir ve solungaç dokusu zebra balığı kullanılan çeşitli hastalık modellerinde sıklıkla incelenir. Bilindiği üzere, deneysel hayvan modeli çalışmalarının sonunda, eğer histopatolojik inceleme yapılacaksa, dokular fikse edilmelidir. Fiksasyonun amacı, hücresel ve hücre dışı bileşenleri mümkün olduğunca in vivo halde korumaktır. Bu nedenle, fiksasyon yöntemlerinin ve fiksatiflerin seçiminin doku takibi üzerinde önemli bir etkisi vardır. Bu çalışmada, zebra balığı solungaç dokusundan ideal preparatlar ve  histolojik görüntüler elde etmek için fiksasyon tekniklerini ve boyama protokollerini optimize etmek amaçlanmıştır. Çalışmamızda, zebra balığı-solungaç dokusundaki ideal histolojiyi belirlemek için, dokular, Bouin, Carnoy, %10’luk nötral tamponlu formaldehit, Davidson ve Dietrich solüsyonları ile fikse edilmiştir. Rutin doku işleminin ardından, kesitler Hematoksilen ve Eosin (H&E) ve Masson Trikrom (MT) boyaları ile boyandı. Sonuç olarak, doku morfolojisi en iyi Bouin ve NBF solüsyonları ile korunmuştur. H&E boyamasında en iyi sonuçlar NBF çözeltisi ile fikse edilmiş dokulardan elde edilirken, Dietrich solüsyonu fiksasyonunun MT boyaması için ideal olduğu bulundu.

Keywords

• Fiksatifler,Hematoxylin ve Eosin,Masson Trikrom,Zebra balığı,Solungaç

References

1. AHMED, H. G., MOHAMMED, A. & HUSSEIN, M. 2011. A comparison study of histochemical staining of various tissues after Carnoy’s and formalin fixation. Sudan Journal of Medical Sciences, 5.
2. ALTURKISTANI, H. A., TASHKANDI, F. M. & MOHAMMEDSALEH, Z. M. 2016. Histological stains: A literature review and case study. Global journal of health science, 8, 72.
3. BANCROFT, J. & GAMBLE, M. 2008. Theory and practice of histological techniques: Elsevier Health Sciences. New York, NY.
4. BANCROFT, J. D. & LAYTON, C. 2018. Connective and other mesenchymal tissues with their stains. Bancroft's Theory and Practice of Histological Techniques E-Book, 153.
5. BENSIMON-BRITO, A., CARDEIRA, J., CANCELA, M. L., HUYSSEUNE, A. & WITTEN, P. E. 2012. Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formation. BMC developmental biology, 12, 28.
6. BIRD, N. C., WINDNER, S. E. & DEVOTO, S. H. 2012. Immunocytochemistry to study myogenesis in zebrafish. Myogenesis. Springer.
7. BRUNDO, M. V. & SALVAGGIO, A. 2018. Zebrafish or Danio rerio: A New Model in Nanotoxicology Study.
8. CARLETON, H. M., DRURY, R. A. B. & WALLINGTON, E. A. 1980. Carleton's histological technique, Oxford University Press, USA.

9. CARSON, F. & HLADIK, C. 2009. Connective and muscle tissue. Histotechnology—A Self Instructional Text, ed. Carson FL, 134-140.
10. CARSON, F. L. 2015. Histotechnology, American Society Of Clini.
11. CHANDRARATHNA, H., NIKAPITIYA, C., DANANJAYA, S., WIJERATHNE, C., WIMALASENA, S., KWUN, H. J., HEO, G.-J., LEE, J. & DE ZOYSA, M. 2018. Outcome of co-infection with opportunistic and multidrug resistant Aeromonas hydrophila and A. veronii in zebrafish: Identification, characterization, pathogenicity and immune responses. Fish & shellfish immunology, 80, 573-581.
12. CHRISTOFFERSEN, T. B., KANIA, P. W., VON GERSDORFF JØRGENSEN, L. & BUCHMANN, K. 2017. Zebrafish Danio rerio as a model to study the immune response against infection with Ichthyophthirius multifiliis. Journal of fish diseases, 40, 847-852.
13. CIMA, L., RIVA, G., D'ERRICO, A., CASARTELLI-LIVIERO, M., CAPELLI, P., TOMEZZOLI, A., MONTIN, U., CARRARO, A., SCARPA, A. & GHIMENTON, C. Fast chromotrope aniline blue special stain is a useful tool to assess fibrosis on liver biopsy during transplantation. Transplantation proceedings, 2017. Elsevier, 667-670.
14. COPPER, J. E., BUDGEON, L. R., FOUTZ, C. A., VAN ROSSUM, D. B., VANSELOW, D. J., HUBLEY, M. J., CLARK, D. P., MANDRELL, D. T. & CHENG, K. C. 2018. Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 208, 38-46.
15. CULLING, C. F. A. 2013. Handbook of histopathological and histochemical techniques: including museum techniques, Butterworth-Heinemann.
16. ÇAKıCı, Ö. & ÜÇÜNCÜ, S. İ. 2007. Zebra balığında Danio rerio'da (Teleostei: Cyprinidae) oosit gelişimi. Ege Journal of Fisheries and Aquatic Sciences, 24.
17. DIETRICH, D. & KRIEGER, H. O. 2009. Histological analysis of endocrine disruptive effects in small laboratory fish, John Wiley & Sons.
18. DOAA, M. M. & HANAN, H. 2013. Histological changes in selected organs of Oreochromis niloticus exposed to doses of lead acetate. J Life Sci Biomed, 3, 256-263.
19. ELTOUM, I., FREDENBURGH, J., MYERS, R. B. & GRIZZLE, W. E. 2001. Introduction to the theory and practice of fixation of tissues. Journal of Histotechnology, 24, 173-190.
20. FEIST, S. W., LANG, T., STENTIFORD, G. & KÖHLER, A. 2004. Biological effects of contaminants: use of liver pathology of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring, International Council for the Exploration of the Sea.
21. FLORES-LOPES, F. & THOMAZ, A. 2011. Histopathologic alterations observed in fish gills as a tool in environmental monitoring. Brazilian Journal of Biology, 71, 179-188.
22. FOURNIE, J. W., KROL, R. M. & HAWKINS, W. E. 2000. Fixation of fish tissues. The laboratory fish. Elsevier.
23. GENTEN, F. 2009. Atlas of fish histology, CRC Press.
24. GNANAPRAGASAM, V. J. 2010. Unlocking the molecular archive: the emerging use of formalin‐fixed paraffin‐embedded tissue for biomarker research in urological cancer. BJU international, 105, 274-278.
25. GRUNOW, B., KIRCHHOFF, T., LANGE, T., MORITZ, T. & HARZSCH, S. 2015. Histochemistry on vibratome sections of fish tissue: a comparison of fixation and embedding methods. Aquatic Biology, 23, 251-263.
26. HADI, A. & ALWAN, S. 2012. Histopathological changes in gills, liver and kidney of fresh water fish, Tilapia zillii, exposed to aluminum. International Journal of Pharmacy & Life Sciences, 3.
27. HOWARD, D. W., LEWIS, E. J., KELLER, B. J. & SMITH, C. S. 2004. Histological techniques for marine bivalve mollusks and crustaceans.
28. HUITING, L., LAROCHE, F. & FENG, H. 2015. The zebrafish as a tool to cancer drug discovery. Austin journal of pharmacology and therapeutics, 3, 1069.
29. KHAN, R. & THULIN, J. 1991. Influence of pollution on parasites of aquatic animals. Advances in parasitology. Elsevier.
30. KIERNAN, J. 2001. Histological and histochemical methods. Theory and practice. Arnold Editor.
31. KUMAR, G. L. & KIERNAN, J. A. 2010. Education Guide-Special Stains and H & E: Pathology, Dako North America.
32. LATENDRESSE, J. R., WARBRITTION, A. R., JONASSEN, H. & CREASY, D. M. 2002. Fixation of testes and eyes using a modified Davidson's fluid: comparison with Bouin's fluid and conventional Davidson's fluid. Toxicologic pathology, 30, 524-533.
33. LEWBART, G. A. 2011. Invertebrate medicine, John Wiley & Sons.
34. MAO, H., SU, P., QIU, W., HUANG, L., YU, H. & WANG, Y. 2016. The use of Masson's trichrome staining, second harmonic imaging and two‐photon excited fluorescence of collagen in distinguishing intestinal tuberculosis from Crohn's disease. Colorectal Disease, 18, 1172-1178.
35. MARASCHIN, B. J., SILVA, V. P. D., ROCK, L., SUN, H., VISIOLI, F., RADOS, P. V. & ROSIN, M. P. 2017. Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues. Brazilian dental journal, 28, 82-84.
36. MARTINELLO, T., PASCOLI, F., CAPORALE, G., PERAZZI, A., IACOPETTI, I. & PATRUNO, M. 2015. Might the Masson trichrome stain be considered a useful method for categorizing experimental tendon lesions? Histol Histopathol, 30, 963-969.
37. MATTHEWS, M. & VARGA, Z. M. 2012. Anesthesia and euthanasia in zebrafish. ILAR journal, 53, 192-204.
38. MIKI, M., OHISHI, N., NAKAMURA, E., FURUMI, A. & MIZUHASHI, F. 2018. Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin’s fluid or Davidson’s fluid. Journal of Toxicologic Pathology, 2018-0001.
39. MOHSENY, A. & HOGENDOORN, P. 2014. Zebrafish as a model for human osteosarcoma. Current Advances in Osteosarcoma. Springer.
40. MORRIS, J. A. 2009. Zebrafish: a model system to examine the neurodevelopmental basis of schizophrenia. Progress in brain research. Elsevier.
41. MUELLER, C., HARPOLE, M. G. & ESPINA, V. 2017. One-step preservation and decalcification of bony tissue for molecular profiling. Molecular Profiling. Springer.
42. MUTO, A. & KAWAKAMI, K. 2013. Prey capture in zebrafish larvae serves as a model to study cognitive functions. Frontiers in neural circuits, 7, 110.
43. NECHIFOR-BOILĂ, A.-C., LOGHIN, A., VACARIU, V., HALAŢIU, V.-B. & BORDA, A. 2015. The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas. Rom J Morphol Embryol, 56, 759-763.
44. NEWMAN, M., EBRAHIMIE, E. & LARDELLI, M. 2014. Using the zebrafish model for Alzheimer’s disease research. Frontiers in genetics, 5, 189.
45. NUSSLEIN-VOLHARD, C. & DAHM, R. 2002. Zebrafish, Oxford University Press.
46. PEREIRA, M. A., DIAS, A. R., FARAJ, S. F., CIRQUEIRA, C. D. S., TOMITAO, M. T., CARLOS NAHAS, S., RIBEIRO JR, U. & DE MELLO, E. S. 2015. Carnoy's solution is an adequate tissue fixative for routine surgical pathology, preserving cell morphology and molecular integrity. Histopathology, 66, 388-397.
47. PETERSON, T. S., KENT, M. L., FERGUSON, J. A., WATRAL, V. G. & WHIPPS, C. M. 2013. Comparison of Fixatives, Fixation Time, and Severity of Infection on PCR Amplification and Detection of Mycobacterium marinum and Mycobacterium chelonae DNA in Paraffin-Embedded Zebrafish (Danio rerio). Diseases of aquatic organisms, 104, 113.
48. PIKARSKY, E., RONEN, A., ABRAMOWITZ, J., LEVAVI-SIVAN, B., HUTORAN, M., SHAPIRA, Y., STEINITZ, M., PERELBERG, A., SOFFER, D. & KOTLER, M. 2004. Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus. Journal of virology, 78, 9544-9551.
49. PROGATZKY, F., COOK, H. T., LAMB, J. R., BUGEON, L. & DALLMAN, M. J. 2016. Mucosal inflammation at the respiratory interface: a zebrafish model. American Journal of Physiology-Lung Cellular and Molecular Physiology, 310, L551-L561.
50. RAHUNEN, K., RIEPPO, L., LEHENKARI, P., FINNILÄ, M. & SAARAKKALA, S. 2016. Evaluation of quantitativity of histological collagen stains in articular cartilage. Osteoarthritis and Cartilage, 24, S307-S308.
51. ROBERTS, R. J. 2012. Fish pathology, John Wiley & Sons.
52. SAHOTA, P. S., POPP, J. A., HARDISTY, J. F. & GOPINATH, C. 2013. Toxicologic pathology: nonclinical safety assessment, CRC press.
53. SINGHAL, P., SINGH, N. N., SREEDHAR, G., BANERJEE, S., BATRA, M. & GARG, A. 2016. Evaluation of histomorphometric changes in tissue architecture in relation to alteration in fixation protocol–an invitro study. Journal of clinical and diagnostic research: JCDR, 10, ZC28.
54. SOUZA, J. P., BARETTA, J. F., SANTOS, F., PAINO, I. M. & ZUCOLOTTO, V. 2017. Toxicological effects of graphene oxide on adult zebrafish (Danio rerio). Aquatic toxicology, 186, 11-18.
55. SPEILBERG, L., EVENSEN, Ø., BRATBERG, B. & SKJERVE, E. 1993. Evaluation of five different immersion fixatives for light microscopic studies of liver tissue in Atlantic salmon Salmo salar. Diseases of aquatic organisms, 17, 47-55.
56. STEFANOVIĆ, D., SAMARDŽIJA, G., REDŽEK, A., ARNAUT, M., NIKIN, Z. & STEFANOVIĆ, M. 2017. Buffered Romanowsky-Giemsa method for formalin fixed, paraffin embedded sections: taming a traditional stain. Biotechnic & Histochemistry, 92, 299-308.
57. STRZYZEWSKA, E., SZAREK, J. & BABINSKA, I. 2016. Morphologic evaluation of the gills as a tool in the diagnostics of pathological conditions in fish and pollution in the aquatic environment: a review. Veterinární Medicína, 61.
58. UNG, C. Y., LAM, S. H., HLAING, M. M., WINATA, C. L., KORZH, S., MATHAVAN, S. & GONG, Z. 2010. Mercury-induced hepatotoxicity in zebrafish: in vivo mechanistic insights from transcriptome analysis, phenotype anchoring and targeted gene expression validation. Bmc Genomics, 11, 212.
59. USLU, B., DIOGUARDI, C. C., HAYNES, M., MIAO, D.-Q., KURUS, M., HOFFMAN, G. & JOHNSON, J. 2017. Quantifying growing versus non-growing ovarian follicles in the mouse. Journal of ovarian research, 10, 3.
60. WESTERFIELD, M. 2000. The zebrafish book: a guide for the laboratory use of zebrafish. http://zfin. org/zf_info/zfbook/zfbk. html.
61. WILSON, J. M., BUNTE, R. M. & CARTY, A. J. 2009. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, 48, 785-789.