Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies
Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies
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DOI：
10.1369/jhc.2008.950105
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发表时间：
2008-11-01
影响因子：
3.2
通讯作者：
Wernig, Anton
中科院分区：
文献类型：
--
作者：
Mundegar, Rustam R.;Franke, Elke;Wernig, Anton
如果一抗来自与被检组织相同的物种，则间接免疫组织化学方法的抗原检测会受到高背景染色的阻碍。这种高背景可以通过在PBS中煮沸切片而在小鼠骨骼肌的未固定的低温恒温切片中消除，然后可以用鼠单克隆抗体容易地检测几种蛋白质，甚至包括低丰度的肌营养不良蛋白。然而，并非所有抗原都能经受煮沸过程。这些抗原中的一些的免疫反应性可以通过随后在Triton X-100中洗涤来恢复，而其他蛋白质的免疫反应性不能通过这种去污剂处理来恢复。当这样的热不稳定的蛋白质用多克隆一抗标记，然后用二氯三嗪基氨基荧光素缀合的二抗标记并煮沸时，荧光信号持续存在，然后可以用单克隆抗体处理切片，用于不受煮沸影响的蛋白质的双重免疫染色。某些荧光染料在加热时的这种稳定性也可以用于用鼠单克隆抗体对两种不同的热稳定蛋白质进行双重免疫荧光标记，以及用于与Y染色体荧光原位杂交相结合。我们的方法应扩大适用于组织来源于相同物种的单克隆抗体的单克隆抗体的范围。（J Histochem Cytochem 56：969-975，2008）
Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein-conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969-975, 2008)