Garton H J, Schoenwolf G C
Department of Neurosurgery, University of Utah School of Medicine, Salt Lake City 84132, USA.
Anat Rec. 1996 Jan;244(1):112-17. doi: 10.1002/(SICI)1097-0185(199601)244:1<112::AID-AR11>3.0.CO;2-S.
Previously it has been difficult to localize in histological sections fluorescent dyes used to label living cells in early embryos. Fluorescent dyes typically are readily soluble in alcohol, xylene, and other common solvents used for conventional paraffin processing. Consequently, they are lost during paraffin embedment. Loss of label can be circumvented with the use of frozen sections, but this technique is laborious to use with young gastrulating and neurulating embryos and it is difficult to obtain consistently high-quality serial sections. Alternative methods such as photoconversion have been used with the fluorescent carbocyanine dye DiI. In this procedure, a diaminobenzidine (DAB)-insoluble reaction product can be deposited in the tissues of whole embryos (using UV photoconversion), which can later be viewed in conventional paraffin sections, but this method is time intensive, technically demanding, and allows for processing of only a single embryo at a time. Moreover, in our hands photoconversion produces inconsistent results and frequently yields significant nonspecific staining.
We have developed an immunohistochemically based process for demonstrating cells labeled with the fluorescent dye [5- (and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)]. With this new technique, cells labeled in living embryos with CFSE can be viewed after later development, first in whole mounts and subsequently in conventional paraffin serial sections. Typically, selected regions of mouse and chick embryos are injected with dye, cultured for periods of about 24 h, viewed with fluorescence microscopy (and recorded on videotape) using an appropriate filter set, and fixed with a formaldehyde-based fixative. An antifluorescein antibody is next bound to the fluorescein groups of CFSE, and an insoluble reaction product is then generated using a horseradish peroxidase (HRP)-conjugated secondary antibody and DAB.
The DAB reaction product deposited with our novel technique can be seen readily in whole mounts using standard stereo light microscopy, providing a permanent label. Moreover, after routine processing for paraffin embedment and histological serial sectioning, the reaction product persists, allowing detailed analyses of the positions and fates of labeled cells.
This simple, immunocytochemical technique, tested in both mouse and chick embryos, provides a highly specific, permanent, and reproducible method for localizing descendants of fluorescently labeled living cells in conventional paraffin sections.
以往在组织学切片中很难定位用于标记早期胚胎活细胞的荧光染料。荧光染料通常易溶于酒精、二甲苯和其他用于传统石蜡处理的常见溶剂。因此，它们在石蜡包埋过程中会丢失。使用冷冻切片可以避免标记物的丢失，但对于处于原肠胚形成和神经胚形成阶段的幼胚来说，这种技术操作繁琐，且难以 consistently 获得高质量的连续切片。诸如光转换等替代方法已用于荧光碳菁染料 DiI。在此过程中，二氨基联苯胺（DAB）不溶性反应产物可沉积在整个胚胎的组织中（使用紫外光转换），随后可在传统石蜡切片中观察到，但该方法耗时、技术要求高，且一次只能处理单个胚胎。此外，在我们的操作中，光转换产生的结果不一致，且经常产生显著的非特异性染色。
我们开发了一种基于免疫组织化学的方法来显示用荧光染料[5-（及-6）-羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）]标记的细胞。使用这种新技术，在活胚胎中用 CFSE 标记的细胞在后续发育后，首先可以在整装标本中观察，随后可以在传统石蜡连续切片中观察。通常，将染料注射到小鼠和鸡胚胎的选定区域，培养约 24 小时，使用适当的滤光片组通过荧光显微镜观察（并录制在录像带上），然后用基于甲醛的固定剂固定。接下来，抗荧光素抗体与 CFSE 的荧光素基团结合，然后使用辣根过氧化物酶（HRP）偶联的二抗和 DAB 产生不溶性反应产物。
使用我们的新技术沉积的 DAB 反应产物在使用标准立体光学显微镜的整装标本中很容易看到，提供了一个永久性标记。此外，在进行石蜡包埋和组织学连续切片的常规处理后，反应产物仍然存在，从而可以对标记细胞的位置和命运进行详细分析。
这种简单的免疫细胞化学技术在小鼠和鸡胚胎中都进行了测试，为在传统石蜡切片中定位荧光标记活细胞的后代提供了一种高度特异性、永久性和可重复的方法。
