获取高质量的兔眼全组织冷冻切片。
Obtaining High-Quality Cryosections of Whole Rabbit Eye.
机构信息
Keck School of Medicine, University of Southern California.
The Vision Center, Department of Surgery, Children's Hospital Los Angeles; The Saban Research Institute, Children's Hospital Los Angeles; USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California.
出版信息
J Vis Exp. 2023 Nov 10(201). doi: 10.3791/66115.
This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 °C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.
本方案描述了如何从小型动物（如兔子）中获取高质量的视网膜冷冻切片。眼球摘出后，先短暂浸泡于固定液中。然后去除角膜和虹膜，在 4°C 下再固定过夜。固定后，移除晶状体。接着将眼球置于冷冻模具中并用包埋介质填充。通过移除晶状体，包埋介质可以更好地接触玻璃体，从而使视网膜更稳定。重要的是，眼球应在包埋介质中孵育过夜，以确保玻璃体完全渗透。过夜孵育后，眼球在干冰上冷冻并切片。可以获得整个视网膜切片，用于免疫组织化学。可以使用标准染色方案来研究抗原在视网膜组织中的定位。遵循本方案可获得高质量的视网膜冷冻切片，可用于任何免疫组织化学实验。
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