Related Experiment Video

Updated: May 24, 2026

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation
10:21

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Published on: December 25, 2017

Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

Meike M Mostegl1, Barbara Richter, Nora Dinhopl

  • 1Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
|February 25, 2012
PubMed

Insights

Prolonged formalin fixation impacts in situ hybridization (ISH) signal intensity for detecting pathogens. Increased proteinase K can restore signals, but effectiveness varies by infectious agent and protocol.

Area of Science:

  • Veterinary Pathology
  • Molecular Diagnostics
  • Histopathology

Background:

  • Chromogenic in situ hybridization (ISH) is vital for pathogen detection in diagnostic pathology.
  • Formalin fixation can impede nucleic acid detection in FFPE tissues due to cross-linking.
  • The impact of fixation duration on ISH signal intensity requires further investigation.

Purpose of the Study:

  • To evaluate the effect of formalin fixation time on ISH signal intensity for specific viral and protozoal pathogens.
  • To assess the efficacy of increased proteinase K concentrations in counteracting formalin-induced cross-linking.

Main Methods:

  • Formalin fixation of tissue samples at varying intervals.
  • Pathogen-specific chromogenic in situ hybridization (ISH).
  • Comparison of standard and increased proteinase K concentrations.

Main Results:

  • ISH signal intensity remained stable for extended periods (up to 55 weeks) for Porcine circovirus-2, Porcine respiratory and reproductive virus, Cryptosporidium serpentis, and Tritrichomonas sp.
  • Signal intensity declined after prolonged fixation.
  • A 4-fold increase in proteinase K restored ISH signals to initial levels for all tested agents.

Conclusions:

  • The duration of formalin fixation significantly affects ISH signal intensity, with pathogen-specific variations.
  • Enhanced proteinase K treatment can effectively mitigate formalin-induced cross-linking.
  • Optimization of pretreatment protocols is crucial for reliable ISH-based pathogen detection in FFPE tissues.

More Related Videos

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes11:36

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Published on: January 6, 2015

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization03:55

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Published on: July 12, 2024

Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
Fixation and Sectioning01:03

Fixation and Sectioning

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...
FISH - Fluorescent In-situ Hybridization02:07

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...