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Fixation and Sectioning01:03

Fixation and Sectioning

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Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...
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Related Experiment Video

Updated: Dec 10, 2025

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation
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Direct formalin fixation induces widespread transcriptomic effects in archival tissue samples.

Leah C Wehmas1, Susan D Hester2, Charles E Wood2,3

  • 1Office of Research and Development, U.S. Environmental Protection Agency, MD-B105-03, 109 T.W. Alexander Drive, Research Triangle Park, NC, 27709, USA. wehmas.leah@epa.gov.

Scientific Reports
|September 4, 2020
PubMed
Summary

Formalin fixation introduces RNA sequencing artifacts, primarily down-regulating gene expression. Freezing samples before fixation minimizes these transcriptional changes, preserving genomic integrity for FFPE research.

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Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
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Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Archival formalin-fixed paraffin-embedded (FFPE) tissues are valuable for genomic research.
  • Formalin fixation can introduce artifacts that potentially bias RNA sequencing (RNA-seq) results.
  • Understanding these fixation-induced artifacts is crucial for accurate data interpretation.

Purpose of the Study:

  • To evaluate global changes in RNA-sequencing profiles between matched frozen and FFPE liver samples.
  • To determine the impact of different fixation protocols on transcriptional profiles.
  • To assess whether direct formalin fixation affects chemical exposure response profiles.

Main Methods:

  • RNA-sequencing was performed on mouse liver samples subjected to four conditions: fresh-frozen, direct formalin fixation, frozen then formalin-fixed, and frozen then ethanol-fixed and paraffin-embedded.
  • Differential gene expression analysis was conducted to compare RNA-seq profiles across conditions.
  • Retrospective studies analyzed paired frozen and FFPE samples for pathway enrichment.

Main Results:

  • Direct formalin fixation induced 2,946 differentially expressed genes (DEGs) compared to fresh-frozen samples, with 98% being down-regulated.
  • Freezing samples prior to formalin fixation reduced DEGs by at least 95% compared to direct fixation.
  • Direct fixation enriched for pathways related to oxidative stress, mitochondrial dysfunction, and transcription initiation.
  • Direct formalin fixation did not significantly alter chemical exposure response profiles.

Conclusions:

  • The majority of transcriptional artifacts in FFPE liver samples occur during the formalin fixation process itself.
  • Pre-freezing samples before formalin fixation significantly mitigates fixation-induced transcriptional alterations.
  • FFPE samples remain a valuable resource for genomic research when fixation artifacts are understood and accounted for.