A method to perform Western blots of microscopic areas of histological sections
A method to perform Western blots of microscopic areas of histological sections
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DOI：
10.1369/jhc.5a6818.2006
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发表时间：
2006-05-01
影响因子：
3.2
通讯作者：
Kretzschmar, HA
中科院分区：
文献类型：
--
作者：
Krebs, B;Kohlmannsperger, V;Kretzschmar, HA
蛋白质印迹法是一种强有力的特异性检测蛋白质的研究方法。然而，由于所需的最小体积和预处理，迄今为止几乎不可能研究组织的微观体积。在此，我们描述了一种直接从冷冻或石蜡包埋组织的组织切片进行蛋白质印迹的方法。小鼠脑的小组织学区域通过切片裂解缓冲液裂解，进行小型化SDS-PAGE，并通过免疫印迹检测。因此，可检测到仅相当于小鼠皮层的15个皮层神经元的面积。这提供了相关的组织学结果的可能性，生化调查。此外，酶预处理应用于鉴定朊病毒蛋白的主要切割产物的糖基化。此外，切片裂解缓冲液是保存和研究磷酸化位点的复杂方法，如磷酸化Akt和ERK所示。所提出的技术结合了组织学与蛋白质印迹技术，将是离散的组织领域的调查价值。
Western blotting is one powerful research method to specifically detect proteins. However, it has been barely possible to investigate microscopic volumes of tissue so far because of the required minimum volumes and the pretreatment. Herein, we describe a method of performing Western blots directly from the histological section of frozen or paraffin-embedded tissue. Small histological areas of a mouse brain were lysed by section lysis buffer, subjected to a miniaturized SDS-PAGE, and detected by immunoblotting. Thereby, an area equivalent to only 15 cortical neurons of mouse cortex was detectable. This offers the possibility of correlating histological findings to biochemical investigations. In addition, enzymatic pretreatment was applied to identify the glycosylation of the major cleavage product of the prion protein. Moreover, the section lysis buffer is a sophisticated method to conserve and investigate phosphorylation sites as demonstrated here by phopsphorylated Akt and ERK. The presented technique combines histology with Western blotting techniques and will be of value for investigations of discrete tissue areas.