Moon Il Soo, Cho Sun-Jung, Jin Ingnyol, Walikonis Randall
Department of Anatomy, College of Medicine, Dongguk University, Gyeongju 780-714, Korea.
Mol Cells. 2007 Aug 31;24(1):76-82.
By combining in situ hybridization (ISH) and immunocytochemistry (IC), microscopic topological localization of mRNAs and proteins can be determined. Although this technique can be applied to a variety of tissues, it is particularly important for use on neuronal cells which are morphologically complex and in which specific mRNAs and proteins are located in distinct subcellular domains such as dendrites and dendritic spines. One common technical problem for combined ISH and IC is that the signal for immunocytochemical localization of proteins often becomes much weaker after conducting ISH. In this manuscript, we report a simplified but robust protocol that allows immunocytochemical localization of proteins after ISH. In this protocol, we fix cultured cortical or hippocampal neurons with 4% paraformaldehyde (PFA), rinse briefly in PBS, and then further fix the cells with C methanol. Our method has several major advantages over previously described ones in that (1) it is simple, as it is just consecutive routine fixation procedures, (2) it does not require any special alteration to the fixation procedures such as changes in salt concentration, and (3) it can be used with antibodies that are compatible with either methanol (MeOH-) or PFA-fixed target proteins. To our best knowledge, we are the first to employ this fixation method for fluorescence ISH + IC.
通过将原位杂交（ISH）和免疫细胞化学（IC）相结合，可以确定mRNA和蛋白质的微观拓扑定位。尽管该技术可应用于多种组织，但对于形态复杂且特定mRNA和蛋白质位于不同亚细胞结构域（如树突和树突棘）的神经元细胞而言，它尤为重要。ISH和IC联用的一个常见技术问题是，在进行ISH后，蛋白质免疫细胞化学定位的信号通常会变得弱得多。在本论文中，我们报告了一种简化但可靠的方案，该方案允许在ISH后进行蛋白质的免疫细胞化学定位。在此方案中，我们用4%多聚甲醛（PFA）固定培养的皮质或海马神经元，在PBS中短暂冲洗，然后用冰冷甲醇进一步固定细胞。我们的方法与先前描述的方法相比有几个主要优点：（1）它很简单，因为它只是连续的常规固定程序；（2）它不需要对固定程序进行任何特殊改动，如盐浓度的变化；（3）它可以与与甲醇（MeOH-）或PFA固定的靶蛋白兼容的抗体一起使用。据我们所知，我们是第一个将这种固定方法用于荧光ISH + IC的。
