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Histomorphological and Molecular Assessments of the Fixation Times Comparing Formalin and Ethanol-Based Fixatives.

Authors
  • Chung, Joon-Yong1
  • Song, Joon Seon1, 2
  • Ylaya, Kris1
  • Sears, John D1
  • Choi, Lauren1
  • Cho, Hanbyoul1, 3
  • Rosenberg, Avi Z4
  • Hewitt, Stephen M1, 4
  • 1 Experimental Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
  • 2 Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. , (North Korea)
  • 3 Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea. , (North Korea)
  • 4 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland.
Type
Published Article
Journal
Journal of Histochemistry & Cytochemistry
Publisher
SAGE Publications
Publication Date
Feb 01, 2018
Volume
66
Issue
2
Pages
121–135
Identifiers
DOI: 10.1369/0022155417741467
PMID: 29125916
Source
Medline
Keywords
License
Unknown

Abstract

The lack of standardization of tissue handling and processing hinders the development and validation of new biomarkers in research and clinical settings. We compared the histomorphology and the quality and quantity of biomolecules in paraffin-embedded mouse tissues, followed by fixation with neutral buffered formalin (NBF), 70% ethanol, and buffered ethanol (BE70) fixative. The quality of the histomorphology and immunohistochemistry in BE70 was relatively time-independent, whereas those in NBF rapidly decreased after 1 week of fixation. Protein recovered from tissue fixed in 70% ethanol and BE70 was compatible with Western blot and protein array using AKT and GAPDH antibodies, regardless of the fixation time. In addition, the quality and quantity of RNA extracted from tissue in ethanol-based fixative showed minimal changes from 4 hr to 6 months, whereas NBF had a dramatic detrimental change in RNA quality after 1 week of fixation. Furthermore, ethanol-based fixative offers a superior DNA template for PCR amplification-based molecular assays than NBF. In conclusion, coagulative, ethanol-based fixatives show a broader time spectrum than the aldehyde crosslinking fixative NBF in their histomorphological features and the quantity and quality of the biomolecules from paraffin-embedded tissue, and they may facilitate the use of fixative-fixed paraffin-embedded tissues in research and clinical laboratories, avoiding overfixation.

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