Shindler K S, Roth K A
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Histochem Cytochem. 1996 Nov;44(11):1331-5. doi: 10.1177/44.11.8918908.
Monoclonal antibodies (MAbs) capable of recognizing developmental stage-specific neuronal epitopes are becoming increasingly available. Because most of these MAbs are raised in a single species (mouse), simultaneous immunofluorescent detection of multiple epitopes has been difficult. We have taken advantage of the high sensitivity of tyramide signal amplification to develop a protocol that permits simultaneous detection of two antibodies raised in the same species. One primary antibody was applied at a concentration below the detection limit of fluorescently labeled secondary antibodies, yet sufficient for detection with the tyramide system. This first primary antibody was then effectively neglected during application of a second primary antibody that was detected by conventional fluorescently labeled secondary antibodies. Specifically, dual labeling for nestin and MAP2 was used to distinguish neuronal stem cells and precursor cells from immature postmitotic neurons, and synapsin I and GAP43 immunostaining was used to distinguish neurons with established synaptic connections from developing neurons. We have used this technique for staining both tissue sections and cultured cells from the embryonic mouse brain. This technique should be widely applicable and offers a simple procedure for simultaneously detecting two antigens when antibodies from only a single species are available.
能够识别发育阶段特异性神经元表位的单克隆抗体（MAbs）越来越多。由于这些单克隆抗体大多是在单一物种（小鼠）中产生的，因此同时免疫荧光检测多个表位一直很困难。我们利用酪胺信号放大的高灵敏度开发了一种方案，该方案允许同时检测在同一物种中产生的两种抗体。一种一抗以低于荧光标记二抗检测限的浓度应用，但足以用酪胺系统进行检测。在应用由常规荧光标记二抗检测的第二种一抗时，第一种一抗实际上被忽略了。具体而言，使用巢蛋白和MAP2的双重标记来区分神经干细胞和前体细胞与未成熟的有丝分裂后神经元，使用突触素I和GAP43免疫染色来区分具有已建立突触连接的神经元与发育中的神经元。我们已将此技术用于对来自胚胎小鼠脑的组织切片和培养细胞进行染色。当只有来自单一物种的抗体可用时，该技术应具有广泛的适用性，并为同时检测两种抗原提供了一个简单的程序。
