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Updated: Jul 29, 2025
Histological-Based Stainings Using Free-Floating Tissue Sections
Published on: August 25, 2020
A novel immunohistochemical protocol for paraffin embedded tissue sections using free-floating techniques
Carolina Muniz Partida1, Eric Walters1
1Department of Biochemistry and Molecular Biology, Howard University College of Medicine, Washington, DC, United States.
Insights
We developed a novel free-float immunohistochemistry protocol for paraffin-embedded tissues (PFFP). This method enhances tissue analysis, saving time, resources, and tissue samples.
Area of Science:
- Neuroscience
- Histology
- Biochemistry
Background:
- Immunohistochemistry (IHC) is crucial for visualizing tissue and protein expression.
- Current free-floating IHC methods use fragile cryo- or vibratome sections with poor morphology.
- Limited information exists on free-floating IHC for paraffin-embedded tissues.
Purpose of the Study:
- To develop and validate a free-float immunohistochemistry protocol for paraffin-embedded tissue (PFFP).
- To assess PFFP's efficiency in localizing specific protein markers in various mouse brain regions.
- To demonstrate PFFP's compatibility with different detection methods and downstream applications.
Main Methods:
- Developed a novel free-float protocol for paraffin-embedded tissue (PFFP).
- Applied PFFP to mouse brain sections (hippocampus, olfactory bulb, striatum, cortex).
- Utilized chromogenic DAB and immunofluorescence detection with and without antigen retrieval.
Main Results:
- PFFP successfully localized GFAP, olfactory marker protein, tyrosine hydroxylase, and Nestin.
- Effective antigen localization was achieved with and without antigen retrieval.
- Demonstrated PFFP's adaptability for various detection methods.
Conclusions:
- PFFP offers a time-, resource-, and tissue-saving alternative to traditional IHC methods.
- The PFFP protocol expands the utility of paraffin-embedded tissues for advanced analyses.
- PFFP is compatible with in situ hybridization, protein interaction studies, and pathological diagnosis.
Abstract:
Immunohistochemistry (IHC) is a well-established and widely used protocol used to visualize tissue architecture, protein expression and localization. Free-floating methods for IHC employ tissue sections that are cut from a cryostat or vibratome. The limitations of these tissue sections are tissue fragility, poor morphology, and the need to use sections of 20-50 μm. In addition, there is a void of information regarding the use of free floating immunohistochemical techniques on paraffin embedded tissue. To address this, we developed a free-float IHC protocol with paraffin embedded tissue (PFFP) that saves time, resources, and tissues. PFFP localized GFAP, olfactory marker protein, tyrosine hydroxylase, and Nestin expression in mouse hippocampal, olfactory bulb, striatum, and cortical tissue. Successful localization of these antigens was achieved using PFFP with and without antigen retrieval, with subsequent chromogenic DAB (3,3'-diaminobenzidine) development and immunofluorescence detection methods. The application of the PFFP in combination with methodologies of in situ hybridization, protein/protein interactions, laser capture dissection, and pathological diagnosis expands the versatility of paraffin embedded tissues.
