Bioimaging, The Pirbright Institute, Pirbright, Woking, UK.
Methods Mol Biol. 2022;2503:51-61. doi: 10.1007/978-1-0716-2333-6_3.
The confocal laser scanning microscope allows us to examine tissue sections in greater detail than a widefield fluorescence microscope. However, this requires samples to be better preserved than standard cryostat sections, which are not usually aldehyde-fixed. Thick sections (approximately 70 μm) of formaldehyde-fixed tissue can be cut using a vibrating microtome and subsequently labeled with primary and secondary fluorescent antibodies and/or fluorescent stains. When imaged in the confocal microscope, these samples allow us to collect high-resolution images, detailing the intracellular location of multiple proteins and structures. In this chapter, we describe the technique used to prepare vibrating microtome sections, using porcine tissue infected with African swine fever virus as an example. This technique can easily be applied to any animal tissue with any suitable combination of antibodies, depending on the hypothesis.
共聚焦激光扫描显微镜使我们能够比宽场荧光显微镜更详细地检查组织切片。然而，这需要比标准的冷冻切片更好地保存样本，而标准的冷冻切片通常不是醛固定的。可以使用振动切片机切割厚切片（约 70μm），然后用一抗和/或二抗荧光抗体以及荧光染料进行标记。在共聚焦显微镜中成像时，这些样品使我们能够收集高分辨率图像，详细描述多种蛋白质和结构的细胞内位置。在本章中，我们将描述使用感染非洲猪瘟病毒的猪组织作为示例制备振动切片的技术。根据假设，这种技术可以很容易地应用于任何动物组织，并且可以与任何合适的抗体组合使用。
