Zucker Robert M, Rogers John M
Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, NC, USA.
Methods Mol Biol. 2019;1965:297-311. doi: 10.1007/978-1-4939-9182-2_20.
After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs.
Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction.
Embryos or tissues as thick as 500 microns (μm) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orange staining.
The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500 μm thick, and stained specimens can be fixed and mounted on depression slides.
荧光染料掺入细胞、组织和生物体后，共聚焦显微镜可用于观察三维结构。溶酶体示踪红（LT）是一种可被多聚甲醛固定的探针，它聚集在细胞的酸性区室中，指示溶酶体活性高和吞噬作用的区域，这两者均与凋亡活性相关。因此，LT是共聚焦显微镜下可视化凋亡的良好指标。凋亡细胞死亡的LT染色结果与其他全胚胎凋亡活性染料如硫酸尼罗蓝和中性红密切相关，其额外的好处是可原位固定。硫酸尼罗蓝也可用作非活性、非特异性染料来观察一般形态。诸如吖啶橙之类的染料可用于固定胚胎的表面染色，以产生类似于扫描电子显微镜图像的共聚焦图像。
用LT对小鼠胚胎进行染色，用多聚甲醛/戊二醛固定，用甲醇脱水，并用苄醇/苯甲酸苄酯（BABB）透明化。经过这种处理后，组织几乎变得透明。将胚胎置于凹面载玻片上，通过共聚焦显微镜对连续切片进行成像，然后进行三维重建。
用BABB透明化后，厚度达500微米（μm）的胚胎或组织均可被观察到。溶酶体示踪染色揭示了器官发生期小鼠胚胎中的凋亡区域。通过将自发荧光与固定胚胎的硫酸尼罗蓝染色相结合，或与吖啶橙染色进行不透明表面染色，有助于对组织进行形态学观察。
使用BABB对LT活体染色并固定的胚胎进行透明化处理，可使组织的折射率与悬浮介质相匹配，从而增加共聚焦显微镜下激光的穿透深度。用作非活性染料的硫酸尼罗蓝可对固定胚胎进行非特异性染色，然后可用水杨酸甲酯进行透明化处理以进行共聚焦观察。本文所述的样品制备和染色程序，结合共聚焦激光扫描显微镜的优化，能够检测和观察厚度达500μm胚胎的形态结构和凋亡情况，并且染色标本可固定并置于凹面载玻片上。
