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Detection of constitutive and inducible HSP70 proteins in formalin fixed human brain tissue
A Preusse-Prange1, J-H Modrow1, T Schwark1
1Institute of Legal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany.
Insights
Formalin fixation impacts protein detection in forensic histology. While Western blotting fails with formalin-fixed tissues, immunohistochemistry (IHC) offers reproducible detection of heat shock proteins (HSP70) for extended periods.
Area of Science:
- Forensic histology
- Molecular biology
- Protein analysis
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are crucial for long-term forensic research.
- Formalin's effect on macromolecules can compromise molecular experiment reliability.
- Understanding formalin's impact on protein detection is vital for reproducible results.
Purpose of the Study:
- To assess formalin's negative effects on protein detection methods.
- To quantify the extent of formalin-induced damage on proteins.
- To compare the suitability of Western blotting and immunohistochemistry (IHC) for analyzing formalin-fixed samples.
Main Methods:
- Brain tissue samples were incubated in formalin for up to three months.
- Immunohistochemistry (IHC) was used to detect and quantify heat shock proteins (HSP70 superfamily).
- Western blotting was employed to analyze protein detection reliability and reproducibility.
Main Results:
- Western blot analysis of formalin-fixed tissues yielded unreliable protein detection.
- Reproducible protein detection using immunohistochemistry (IHC) was feasible for up to one month of formalin incubation.
- Formalin significantly degrades proteins, hindering Western blot efficacy.
Conclusions:
- Formalin fixation poses significant challenges for reliable protein analysis, particularly with Western blotting.
- Immunohistochemistry (IHC) remains a viable method for detecting specific proteins in formalin-fixed tissues for limited durations.
- Further research is needed to optimize protocols for molecular analysis of long-term FFPE samples.
Abstract:
The investigation of formalin fixed and paraffin embedded tissue is a routine method in forensic histology. Since these samples are usually stored for decades they provide a unique tissue bank for different scientific issues. In the past, numerous studies were conducted using different kinds of paraffin embedded tissues. However, it is well known that formalin affects macromolecules and thus might hamper reliable and reproducible molecular experiments. The aim of this study was to find out if the treatment with formalin has a negative effect on different protein detection methods and additionally to define the dimension of those possible deleterious effects. We incubated brain tissue samples in formalin for up to three months. After incubation, the samples were analyzed using immunohistochemistry (IHC) and Western blotting to specifically detect and quantify members of the HSP70 superfamily (heat shock proteins). Our study shows that the Western blot analysis of formalin fixed tissues does not allow a reliable detection of proteins at all, while a reproducible detection by IHC was still possible after one month of incubation.
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