Novel tissue preservative and tissue fixative for comparative pathology and animal research
Introduction
Procurement of animal and human tissue is of great importance for biomedical research. Histomorphological evaluation of formalin-fixed, paraffin-embedded tissue stained with hematoxylin and eosin represents common practice in pathology. At the same time, the tissue provides a source of DNA, RNA and proteins (macromolecules) necessary for molecular analysis. Unfortunately, formalin, the most commonly used tissue fixative, does not protect macromolecules (Srinivasan et al., 2002). It degrades RNA and modifies DNA and proteins. Therefore, macromolecules are most commonly extracted from fresh or frozen tissue. However, immediate extraction of macromolecules, particularly during field studies, is impossible in most situations and the storage and transportation of frozen tissue represents a logistical challenge. Furthermore, fresh or frozen tissue has limited value for the assessment of histomorphology or for the performance of ancillary tests. As formalin fixation is of limited value in preserving macromolecules and is also not environmentally friendly, alternative tissue fixatives were tried but with partial success. Recently we have developed a novel tissue fixative and preservative (Vincek et al., 2003). The universal molecular fixative (UMFIX), a mixture of methanol (serving as coagulative fixative) and polyethylene glycol (PEG) (used to decrease excessive nuclear shrinkage), was shown to preserve macromolecules and histomorphology in human tissue at room temperature. It is currently introduced to human clinical practice and is replacing formalin. In this study we tested whether UMFIX can also be used for histomorphological evaluation of animal tissue and whether it preserves RNA at high ambient temperatures so that it could be used as a tissue preservative of macromolecules for animal field studies.
Section snippets
Tissue collection
Mouse, cat, dog, pigeon, rabbit and rat tissue was obtained as residual animal tissue through the approved program for the use of residual animal tissue by the Animal Research Committee at University of Miami. Ant (Camponotus floriadanus), beetle (Phylophaga), earthworm (Lumbricus terrestris) and lizard (Anolis sagrei sagrei) were collected during a field trip.
Tissue fixation
Tissue was fixed in neutral-buffered 10% formalin or UMFIX (Sakura Finetek USA, Inc.) for a period of time ranging from 24 h to 7 days.
Results
To assess histomorphology of animal tissue fixed in our novel fixative we have fixed all major organs of cat, dog, mouse, pigeon, rabbit and rat for 24 h or 7 days in UMFIX at room temperature or environmental temperature (30–34 °C). We have also fixed the same tissue in formalin for 24 h or 7 days at room temperature for comparison. We found that UMFIX preserved tissue, fixed for 1 day or 1 week, shows similar histomorphology regardless of the type of the tissue processor used. Fixation in UMFIX
Discussion
The time-honored processes of formalin fixation and routine overnight tissue processing form the cornerstones of human and veterinary pathology but are of limited value in preserving intact RNA, protein and even DNA. It has, with minor modifications, been used for over 100 years (Blum, 1893). From the beginning, histologists pointed to various problems with formalin. It causes various tissue artifacts and some regarded formalin unsuitable for visualization of nuclear structures, microscopy of
References (32)
- et al.
Using Kryofix as alternative for formalin results in more optimal and standardized immunostaining of paraffin sections
Path. Res. Pract.
(1992) - et al.
Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue
Kidney Int.
(2002) - et al.
Preservation of RNA for functional genomic studies: a multidisciplinary tumor bank protocol
Mod. Pathol.
(2001) - et al.
Evaluation of non-formalin tissue fixation for molecular profiling studies
Am. J. Pathol.
(2002) - et al.
Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using nuclease quantitative reverse transcription-polymerase chain reaction
J. Mol. Diagn.
(2000) - et al.
Expression profiling of mouse endometrial cancers microdissected from ethanol-fixed, paraffin-embedded tissues
Am. J. Pathol.
(2003) - et al.
Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies
Methods
(2001) - et al.
HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues
Pathol. Res. Pract.
(2001) - et al.
Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue
Am. J. Pathol.
(2001) - et al.
Effect of fixatives and tissue processing on the content and integrity of nucleic acids
Am. J. Pathol.
(2002)
A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples
Lab. Invest.
Zinc-based fixative improves preservation of genomic DNA and proteins in histoprocessing
Lab. Invest.
Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications
Proteomics
American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning
J. Toxicol. Clin. Toxicol.
Der formaldehyd als hartungsmittel
Z. Wiss. Microsc.
Establishment of the formalin-free surgical pathology laboratory. Utility of an alcohol-base fixative
Arch. Pathol. Lab. Med.
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