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Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers
Published on: December 31, 2016
Development of a highly sensitive immunohistochemical method to detect neurochemical molecules in formalin-fixed and
Satoshi Goto1, Ryoma Morigaki1, Shinya Okita2
1Department of Motor Neuroscience and Neurotherapeutics, Institute of Health Biosciences, Graduate School of Medical Sciences, Tokushima University Tokushima, Japan ; Parkinson's Disease and Dystonia Research Center, Tokushima University Hospital, Tokushima University Tokushima, Japan.
Insights
A new immunohistochemistry (IHC) method, PBTA, significantly enhances neurochemical antigen detection. This highly sensitive method improves visualization of trace antigens in human brain tissues.
Area of Science:
- Neuroscience
- Biochemistry
- Immunology
Background:
- Immunohistochemistry (IHC) is crucial for identifying neurochemical molecules.
- Current IHC methods have limitations in sensitivity for detecting low-abundance antigens.
Purpose of the Study:
- To develop a highly sensitive immunostaining method for neurochemical antigens.
- To evaluate the efficacy of the novel PBTA method in detecting antigens in human brain tissues.
Main Methods:
- Developed the Polymer-Avidin-Biotin-Tyramide Amplification (PBTA) method, a hybrid protocol.
- Combined polymer, avidin-biotin-complex (ABC), and biotin-tyramide amplification techniques.
- Tested PBTA using [Met]-enkephalin as a target antigen in formalin-fixed, paraffin-embedded human brain tissues.
Main Results:
- PBTA method demonstrated over 100-fold higher sensitivity for IHC compared to polymer and ABC methods.
- PBTA showed approximately 1,000-fold higher sensitivity for enzyme-linked immunosorbent assay (ELISA) compared to ABC.
- Achieved high sensitivity without increasing non-specific background activity in chromogenic and fluorescence detection.
Conclusions:
- The PBTA method offers a significant advancement in IHC sensitivity for neurochemical antigens.
- This technique is effective for visualizing trace amounts of neurochemical peptides and proteins in human brain samples.
- PBTA is a powerful tool for neurochemical research and diagnostics using autopsied brain tissues.
Abstract:
Immunohistochemistry (IHC) is a valuable method for identifying discrete neurochemical molecules by the interaction of target antigens with validated antibodies tagged with a visible label (e.g., peroxidase). We have developed an immunostaining method that is highly sensitive in detection of neurochemical antigens. Our IHC method, which we call the PBTA method, involves a hybrid protocol that implements aspects of both the polymer and avidin-biotin-complex (ABC) methods in combination with biotin-tyramide amplification. When using [Met]-enkephalin as a target antigen, the sensitivity of the PBTA method for IHC was more than 100-fold higher compared with the polymer and ABC methods. In addition, its sensitivity for enzyme-linked immunosorbent assay was about 1,000-fold higher compared with the ABC method. We examined the utility of our IHC method for both chromogenic and fluorescence detection systems used to visualize neurochemical peptides and proteins in formalin-fixed, paraffin-embedded tissues from autopsied human brains. The results convincingly demonstrate that under optimal conditions, our IHC method is highly sensitive without increasing non-specific background activities. Our IHC method could be a powerful tool for detection and visualization of neurochemical antigens that are present even in trace amounts in autopsied human brains.
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