open access

Vol 78, No 2 (2019)
Original article
Submitted: 2018-08-16
Accepted: 2018-10-03
Published online: 2018-10-16
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The effect of prolonged formalin fixation on the staining characteristics of archival human brain tissue

A. Alrafiah12, R. Alshali32
·
Pubmed: 30371933
·
Folia Morphol 2019;78(2):230-236.
Affiliations
  1. Department of Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
  2. Neuroscience Research Unit, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
  3. Department of Anatomy, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

open access

Vol 78, No 2 (2019)
ORIGINAL ARTICLES
Submitted: 2018-08-16
Accepted: 2018-10-03
Published online: 2018-10-16

Abstract

Background: Neurodegenerative disorders include wide range of conditions, which affect millions of people worldwide. Unfortunately, they are incurable and irreversibly progressive. Immunohistochemical staining of paraffin-fixed tissues for both diagnostic and research purposes are widely used. However, large amount of brain tissues are fixed but little is known about whether they are suitable for retrospective studies. The study aimed at investigating the effects of prolonged formalin fixation time on immunohistochemical expression of some common neurodegenerative markers in archival brain specimens. 

Materials and methods: Twenty brain specimens were obtained from hu- man cadavers in the Anatomy Department of King Abdulaziz University that were prefixed in 10% formalin. They were divided into two equal groups according to time of fixation: group 1 — less than 1 year, group 2 — up to 20 years. Histological examination of white and grey matter was done using haematoxylin and eosin, luxol fast blue (LFB) for myelin staining, Con- go red for amyloid plaques, CD 68 for microglial cells, tenascin-C (large ex- tracellular matrix glycoprotein) and caspase 3 antibody for apoptotic cells. 

Results: For both groups, corpus callosum sections displayed myelination with LFB staining. The distribution of CD 68 positive microglial cells was evi- dent in frontal and temporal grey matter, but not in corpus callosum sections. Strongly positive masses were seen in Congo red-stained frontal and temporal sections. Anti-caspase 3 immunostaining revealed positively stained neurons.

Conclusions: Histological and immunohistochemical techniques yielded repro- ducible staining results when applied to human brain tissue stored in formalin for long periods; so they can be used in well preserved biobank material which are the most targeting research areas in neuropathology. 

Abstract

Background: Neurodegenerative disorders include wide range of conditions, which affect millions of people worldwide. Unfortunately, they are incurable and irreversibly progressive. Immunohistochemical staining of paraffin-fixed tissues for both diagnostic and research purposes are widely used. However, large amount of brain tissues are fixed but little is known about whether they are suitable for retrospective studies. The study aimed at investigating the effects of prolonged formalin fixation time on immunohistochemical expression of some common neurodegenerative markers in archival brain specimens. 

Materials and methods: Twenty brain specimens were obtained from hu- man cadavers in the Anatomy Department of King Abdulaziz University that were prefixed in 10% formalin. They were divided into two equal groups according to time of fixation: group 1 — less than 1 year, group 2 — up to 20 years. Histological examination of white and grey matter was done using haematoxylin and eosin, luxol fast blue (LFB) for myelin staining, Con- go red for amyloid plaques, CD 68 for microglial cells, tenascin-C (large ex- tracellular matrix glycoprotein) and caspase 3 antibody for apoptotic cells. 

Results: For both groups, corpus callosum sections displayed myelination with LFB staining. The distribution of CD 68 positive microglial cells was evi- dent in frontal and temporal grey matter, but not in corpus callosum sections. Strongly positive masses were seen in Congo red-stained frontal and temporal sections. Anti-caspase 3 immunostaining revealed positively stained neurons.

Conclusions: Histological and immunohistochemical techniques yielded repro- ducible staining results when applied to human brain tissue stored in formalin for long periods; so they can be used in well preserved biobank material which are the most targeting research areas in neuropathology. 

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Keywords

human brain; immunohistochemistry; formalin fixed; neurodegenerative markers

About this article
Title

The effect of prolonged formalin fixation on the staining characteristics of archival human brain tissue

Journal

Folia Morphologica

Issue

Vol 78, No 2 (2019)

Article type

Original article

Pages

230-236

Published online

2018-10-16

Page views

2637

Article views/downloads

4534

DOI

10.5603/FM.a2018.0099

Pubmed

30371933

Bibliographic record

Folia Morphol 2019;78(2):230-236.

Keywords

human brain
immunohistochemistry
formalin fixed
neurodegenerative markers

Authors

A. Alrafiah
R. Alshali

References (31)
  1. Alafuzoff I, Pikkarainen M, Al-Sarraj S, et al. Interlaboratory comparison of assessments of Alzheimer disease-related lesions: a study of the BrainNet Europe Consortium. J Neuropathol Exp Neurol. 2006; 65(8): 740–757.
  2. Alturkistani HA, Tashkandi FM, Mohammedsaleh ZM. Histological Stains: A Literature Review and Case Study. Glob J Health Sci. 2015; 8(3): 72–79.
  3. Ando Y, Haraoka K, Terazaki H, et al. A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo. Lab Invest. 2003; 83(12): 1751–1759.
  4. Arber D. Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers. Appl Immunohistochem Mol Morphol. 2002; 10(2): 183–186.
  5. Carriel V, Campos A, Alaminos M, et al. Staining Methods for Normal and Regenerative Myelin in the Nervous System. Methods Mol Biol. 2017; 1560: 207–218.
  6. Clement CG, Truong LD. An evaluation of congo red fluorescence for the diagnosis of amyloidosis. Hum Pathol. 2014; 45(8): 1766–1772.
  7. Evers P, Uylings H. An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods . 1997; 72(2): 197–207.
  8. Fox CH, Johnson FB, Whiting J, et al. Formaldehyde fixation. J Histochem Cytochem. 1985; 33(8): 845–853.
  9. Grillo F, Pigozzi S, Ceriolo P, et al. Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections. Histochem Cell Biol. 2015; 144(1): 93–99.
  10. Herold-Mende C, Mueller MM, Bonsanto MM, et al. Clinical impact and functional aspects of tenascin-C expression during glioma progression. Int J Cancer. 2002; 98(3): 362–369.
  11. Holness CL, Simmons DL. Molecular cloning of cd68, a human macrophage marker related to lysosomal glycoproteins. Blood. 1993; 81(6): 1607–1613.
  12. Klüver H, Barrera E. A Method for the Combined Staining of Cells and Fibers in the Nervous System. J Neuropathol Exp Neurol. 1953; 12(4): 400–403.
  13. Lee Y, McKinnon PJ. Detection of apoptosis in the central nervous system. Methods Mol Biol. 2009; 559: 273–282.
  14. Leins A, Riva P, Lindstedt R, et al. Expression of tenascin-C in various human brain tumors and its relevance for survival in patients with astrocytoma. Cancer. 2003; 98(11): 2430–2439.
  15. Machaalani R, Radford JL, Waters KA. Tissue fixation effects on immunohistochemical staining of caspase-3 in brain tissue. Appl Immunohistochem Mol Morphol. 2007; 15(4): 463–470.
  16. Mason JT, O'Leary TJ. Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation. J Histochem Cytochem. 1991; 39(2): 225–229.
  17. Midwood KS, Orend G. The role of tenascin-C in tissue injury and tumorigenesis. J Cell Commun Signal. 2009; 3(3-4): 287–310.
  18. Midwood KS, Hussenet T, Langlois B, et al. Advances in tenascin-C biology. Cell Mol Life Sci. 2011; 68(19): 3175–3199.
  19. Pikkarainen M, Martikainen P, Alafuzoff I. The effect of prolonged fixation time on immunohistochemical staining of common neurodegenerative disease markers. J Neuropathol Exp Neurol. 2010; 69(1): 40–52.
  20. Ploeger S, Guldemond JM, Feirabend HK, et al. Acidification of human brains stored in fixatives. Eur J Morphol. 1993; 31(4): 286–290.
  21. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 1999; 6(2): 99–104.
  22. Sheaffer S, Rosoklija G, Dwork AJ. Myelin staining of archival brain tissue. Clin Neuropathol. 1999; 18(6): 313–317.
  23. Smith EE. Cerebral amyloid angiopathy as a cause of neurodegeneration. J Neurochem. 2018; 144(5): 651–658.
  24. Stankov A, Belakaposka-Srpanova V, Bitoljanu N, et al. Visualisation of Microglia with the use of Immunohistochemical Double Staining Method for CD-68 and Iba-1 of Cerebral Tissue Samples in Cases of Brain Contusions. Pril (Makedon Akad Nauk Umet Odd Med Nauki). 2015; 36(2): 141–145.
  25. Sun Y, Xu Y, Geng L. Caspase-3 inhibitor prevents the apoptosis of brain tissue in rats with acute cerebral infarction. Exp Ther Med. 2015; 10(1): 133–138.
  26. Walker DG, Lue LF. Immune phenotypes of microglia in human neurodegenerative disease: challenges to detecting microglial polarization in human brains. Alzheimers Res Ther. 2015; 7(1): 56.
  27. Webster JD, Miller MA, Dusold D, et al. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem. 2009; 57(8): 753–761.
  28. Weickenmeier J, de Rooij R, Budday S, et al. Brain stiffness increases with myelin content. Acta Biomater. 2016; 42: 265–272.
  29. Wiese S, Karus M, Faissner A. Astrocytes as a source for extracellular matrix molecules and cytokines. Front Pharmacol. 2012; 3: 120.
  30. Wilcock DM, Gordon MN. Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain. Nat Protoc. 2006; 1(3): 1591–1595.
  31. Williams JH, Mepham BL, Wright DH. Tissue preparation for immunocytochemistry. J Clin Pathol. 1997; 50(5): 422–428.

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