Zucker Robert M, Jeffay Susan C
National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC, USA.
Cytometry A. 2006 Aug 1;69(8):930-9. doi: 10.1002/cyto.a.20315.
Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy.
Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths.
Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image.
Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.
卵巢由众多处于不同发育阶段的卵泡、卵母细胞和颗粒细胞组成。这些卵泡中的许多在动物的一生中会经历凋亡过程。通过使用适当的组织制备方法，可以利用三维共聚焦显微镜观察整个卵巢内的事件。
将整个卵巢用溶酶体示踪红（LT）染色，用4%多聚甲醛（PF）和1%戊二醛（Glut）固定，用甲醇（MEOH）脱水，并用苯甲醇和苯甲酸苄酯（BABB）透明处理。使用这种组织制备技术，卵巢变得相对透明，从而可以用共聚焦显微镜观察其形态。利用位于传统显微镜上的光谱成像系统（PARISS）来解读不同激发波长下组织中LT染料光谱和固定产物。
卵泡中的凋亡表现为紧邻卵母细胞的强染色颗粒细胞簇。用Glut和PF固定可保留形态细节，增加组织荧光，从而提高背景图像的信噪比。
经过适当染色、醛固定和BABB透明处理后的厚组织可以成像。LT可使卵泡和核仁中的单个细胞或凋亡细胞簇强烈染色。作为凋亡指标的LT与Glut-PF固定之间的光谱差异被用于观察卵巢形态和凋亡。PARISS分光光度计显示LT在609.6 nm处有光谱峰，Glut-PF在471.3 nm处有光谱峰。正确使用这些荧光分子的光谱是高质量凋亡形态图像的基础。通过用488 nm激光和543/568 nm激光依次对这两种探针成像，荧光串扰减少，图像质量提高。
