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Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy
Published on: September 3, 2017
Reduction of autofluorescence at the microelectrode-cortical tissue interface improves antibody detection
Kelsey A Potter1, Joel S Simon, Bharath Velagapudi
1Department of Biomedical Engineering, Case Western Reserve University, 2071 Martin Luther King Jr. Drive, Wickenden Bldg, Cleveland, OH 44106, USA.
Insights
Copper sulfate treatment significantly reduces autofluorescence from hemosiderin-laden macrophages in immunohistochemistry (IHC). This method improves the detection of low-level inflammatory markers at the microelectrode-cortical tissue interface.
Area of Science:
- Neuroscience
- Biomedical Engineering
- Immunology
Background:
- Immunohistochemistry (IHC) is crucial for detecting neuroinflammation at the microelectrode-cortical tissue interface.
- Autofluorescence, particularly from hemosiderin-laden macrophages (HLMs), complicates accurate signal quantification in IHC.
- HLM autofluorescence interferes with the detection of low-abundance inflammatory markers.
Purpose of the Study:
- To investigate copper sulfate as a method to reduce HLM-derived autofluorescence in IHC.
- To assess the impact of copper sulfate treatment on the detection of both highly and weakly expressed antigens.
- To improve the accuracy and reproducibility of inflammatory marker quantification at the tissue-device interface.
Main Methods:
- Cortical tissue was treated with a minimum of 0.5mM copper sulfate for at least 10 minutes.
- Autofluorescence levels in green and red fluorescent channels were measured before and after treatment.
- The effect of copper sulfate treatment on the quantification of glial fibrillar acidic protein (GFAP) and other weakly expressed antigens was evaluated.
Main Results:
- Copper sulfate treatment reduced native HLM autofluorescence by at least 70% in both fluorescent channels.
- While background signal decreased, copper sulfate treatment did not significantly alter the quantification of highly expressed antigens like GFAP.
- Detection efficiency for weakly expressed antigens at the interface was substantially enhanced by copper sulfate treatment.
Conclusions:
- Copper sulfate incubation is an effective method to significantly reduce HLM-derived autofluorescence during IHC.
- This treatment enables more accurate detection and quantification of faintly expressed inflammatory markers.
- The findings support the use of copper sulfate in IHC protocols for improved analysis of the neuro-inflammatory response at the device-tissue interface.
Abstract:
Immunohistochemistry (IHC) remains among the most utilized methods for detection of inflammatory events occurring at the microelectrode-cortical tissue interface. It has further become a standard protocol to quantify the intensity of this resulting fluorescent signal, normalized to "background", as a measurement of the extent of inflammatory events. Unfortunately, several sources of autofluorescence could result in variations in this user-defined "background". Notably, we found that the presence of hemosiderin-laden macrophages (HLMs) at the interface resulted in a variable source of background in both green and red fluorescent channels. The HLM-derived autofluorescence prevented the reproducible detection of presumably low-level antigens at the interface. Here we show that treatment of the native cortical tissue for no less than 10 min, with a minimum of 0.5mM copper sulfate, resulted in at least a 70% reduction in native HLM autofluorescence in both green and red fluorescent channels. In the case of highly expressed antigens, such as glial fibrillar acidic protein (GFAP), treatment of immuno-labeled tissue with copper sulfate reduced tissue background, compared to standard IHC methodology, but did not result in significant differences in the quantification of normalized signal intensity. However, treatment with copper sulfate substantially enhanced the detection efficiency of weakly expressed antigens at the device-tissue interface. This study demonstrates that the inclusion of copper sulfate incubation during IHC tissue preparation significantly reduced HLM-derived autofluorescence, and allowed for more accurate detection and quantification of faintly expressed inflammatory markers at the device-tissue interface.
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