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Updated: Aug 31, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
Effect of formalin tissue fixation and processing on immunohistochemistry
1Institut für Allgemeine Pathologie und Pathologische Anatomie, Klinikum rechts der Isar, Technische Universität München, Germany. martin.werner@lrz.tum.de
Insights
Standardizing immunohistochemistry requires optimizing tissue fixation and processing. Prompt fixation and controlled duration, alongside fresh processing solutions, can improve staining reproducibility in pathology labs.
Area of Science:
- Pathology
- Laboratory Medicine
- Histology
Background:
- Immunohistochemistry (IHC) staining reproducibility is often compromised by variations in tissue handling.
- Tissue fixation and processing are identified as primary sources of variability in IHC assays.
Purpose of the Study:
- To address the lack of standardization in immunohistochemistry (IHC) staining.
- To identify and propose solutions for variability introduced by tissue fixation and processing.
Main Methods:
- The report summarizes findings from the International Consensus Group on Standardization and Quality Control (ICGSQC) meeting.
- Key issues discussed include formalin fixation challenges and tissue dehydration during processing.
Main Results:
- Formalin fixation variability arises from delayed fixation and inconsistent duration (over- or under-fixation).
- Inadequate tissue dehydration before paraffin embedding is a significant issue in tissue processing.
- Implementing timely fixation (within 30 minutes) and controlled fixation duration (24-48 hours) are recommended.
- Fresh preparation of processing solutions weekly is crucial for preventing dehydration issues.
Conclusions:
- Standardizing tissue fixation and processing is essential for improving immunohistochemistry reproducibility.
- Adherence to recommended fixation and processing protocols can mitigate major sources of IHC staining variation.
Abstract:
Although immunohistochemistry is routinely performed by many pathology laboratories, its standardization still lags behind. A major cause of variation in the reproducibility of immunohistochemical staining is induced by tissue fixation and, to a lesser degree, tissue processing. This report, stemming from the first meeting of the International Consensus Group on Standardization and Quality Control (ICGSQC) in Nice, France, summarizes the problem and suggests solutions to begin to achieve standardization of fixation and processing. Most laboratories use neutral-buffered formalin (10%) for tissue fixation which introduces cross-links, whereas coagulative fixatives are less popular. Problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly. Solutions to these problems could be to start fixation soon (<30 min) after surgical removal of the tissue and to avoid overfixation (>24-48 hrs). For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all solutions freshly every week, depending on the volume of tissue processed. If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains.
