Prehn Anna, Hobusch Constance, Härtig Wolfgang, Michalski Dominik, Krueger Martin, Flachmeyer Bianca
Institute of Anatomy, Leipzig University, Leipzig, Germany.
Paul Flechsig Institute of Brain Research, Leipzig University, Leipzig, Germany.
Front Cell Neurosci. 2023 Jun 2;17:1183232. doi: 10.3389/fncel.2023.1183232. eCollection 2023.
In the setting of stroke, ischemia not only impairs neuronal function, but also detrimentally affects the different components of the neurovascular unit, which are shown to be involved in the transition from reversible to long-lasting tissue damage. In this context, the glial proteins myelin basic protein (MBP) and the 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) as well as the vasculature-associated basement membrane proteins laminin and collagen IV have been identified as ischemia-sensitive elements. However, available data from immunofluorescence and Western blot analyses are often found to be contradictory, which renders interpretation of the respective data rather difficult. Therefore, the present study investigates the impact of tissue pre-treatment and antibody clonality on immunofluorescence measurements of the mentioned proteins in a highly reproducible model of permanent middle cerebral artery occlusion. Here, immunofluorescence labeling using polyclonal antibodies revealed an increased immunofluorescence intensity of MBP, CNP, laminin and collagen IV in ischemic areas, although Western blot analyses did not reveal increased protein levels. Importantly, contrary to polyclonal antibodies, monoclonal ones did not provide increased fluorescence intensities in ischemic areas. Further, we were able to demonstrate that different ways of tissue pre-treatment including paraformaldehyde fixation and antigen retrieval may not only impact on fluorescence intensity measurements in general, but rather one-sidedly affect either ischemic or unaffected tissue. Therefore, immunofluorescence intensity measurements do not necessarily correlate with the actual protein levels, especially in ischemia-affected tissue and should always be complemented by different techniques to enhance reproducibility and to hopefully overcome the translational roadblock from bench to bedside.
在中风的情况下，缺血不仅会损害神经元功能，还会对神经血管单元的不同组成部分产生不利影响，这些组成部分被证明参与了从可逆性组织损伤到持久性组织损伤的转变。在这种情况下，已确定神经胶质蛋白髓鞘碱性蛋白（MBP）和2'，3'-环核苷酸3'-磷酸二酯酶（CNP）以及与血管相关的基底膜蛋白层粘连蛋白和IV型胶原是对缺血敏感的成分。然而，免疫荧光和蛋白质印迹分析的现有数据常常相互矛盾，这使得对各自数据的解释相当困难。因此，本研究在高度可重复的大脑中动脉永久性闭塞模型中，研究了组织预处理和抗体克隆性对上述蛋白质免疫荧光测量的影响。在这里，使用多克隆抗体的免疫荧光标记显示，缺血区域中MBP、CNP、层粘连蛋白和IV型胶原的免疫荧光强度增加，尽管蛋白质印迹分析未显示蛋白质水平升高。重要的是，与多克隆抗体相反，单克隆抗体在缺血区域并未提供增强的荧光强度。此外，我们能够证明，包括多聚甲醛固定和抗原修复在内的不同组织预处理方式不仅可能总体上影响荧光强度测量，而且可能单方面影响缺血或未受影响的组织。因此，免疫荧光强度测量不一定与实际蛋白质水平相关，尤其是在受缺血影响的组织中，并且应该始终辅以不同技术以提高可重复性，并有望克服从实验台到临床应用的转化障碍。
