Reduction of autofluorescence at the microelectrode-cortical tissue interface improves antibody detection
Reduction of autofluorescence at the microelectrode-cortical tissue interface improves antibody detection
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DOI：
10.1016/j.jneumeth.2011.09.024
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发表时间：
2012-01-15
影响因子：
3
通讯作者：
Capadona, Jeffrey R.
中科院分区：
文献类型：
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作者：
Potter, Kelsey A.;Simon, Joel S.;Capadona, Jeffrey R.
免疫组织化学（IHC）仍然是用于检测在微电极-皮质组织界面处发生的炎性事件的最常用的方法之一。它已进一步成为一种标准方案，以量化这种所得荧光信号的强度，标准化为“背景”，作为炎症事件程度的测量。不幸的是，几种自发荧光源可能导致用户定义的“背景”发生变化。值得注意的是，我们发现，含铁血黄素的巨噬细胞（HLM）在界面处的存在导致在绿色和红色荧光通道的背景的可变来源。HLM衍生的自发荧光阻止了界面处推测为低水平抗原的可重复检测。在这里，我们表明，治疗的天然皮质组织不少于10分钟，与最低的0.5 mM硫酸铜，导致至少70%的天然HLM自体荧光减少在两个绿色和红色荧光通道。在高度表达的抗原，如胶质纤维酸性蛋白（GFAP）的情况下，与标准的IHC方法相比，用硫酸铜处理免疫标记的组织减少了组织背景，但没有导致归一化信号强度定量的显著差异。然而，用硫酸铜处理显著增强了装置-组织界面处弱表达抗原的检测效率。本研究表明，在IHC组织制备过程中加入硫酸铜孵育可显著降低HLM衍生的自体荧光，并可更准确地检测和定量器械-组织界面处微弱表达的炎症标志物。由爱思唯尔公司出版
lmmunohistochemistry (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 0.5 mM 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. Published by Elsevier B.V.