Reduction of autofluorescence at the microelectrode–cortical tissue interface improves antibody detection

• Published in: Journal of neuroscience methods 2012-01, Vol.203 (1), p.96-105
• Main Authors: Potter, Kelsey A., Simon, Joel S., Velagapudi, Bharath, Capadona, Jeffrey R.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Autofluorescence; Cerebral Cortex - immunology; Cerebral Cortex - pathology; Copper Sulfate; Electrodes, Implanted - adverse effects; Hemosiderin - chemistry; Hemosiderin-laden macrophage; Image Processing, Computer-Assisted; Immunohistochemistry; Immunohistochemistry - methods; Inflammation - etiology; Inflammation - immunology; Inflammation - pathology; Macrophages - chemistry; Macrophages - metabolism; Male; Microelectrodes - adverse effects; Microscopy, Fluorescence - methods; Neural electrode; Rats; Rats, Sprague-Dawley; Signal-To-Noise Ratio
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2011.09.024

[Display omitted] ► Hemosiderin-laden cells at the electrode–tissue interface autofluorescence in both green and red. ► Incubation in copper sulfate significantly reduced hemosiderin-derived autofluorescence. ► Copper sulfate treatment provided no advantage for the detection of highly expressed antigens. ► Copper sulfate treatment enhanced the detection resolution between weakly expressed antigens. 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 10min, 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.