Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.
Department of Urology, Aarhus University Hospital, Aarhus, Denmark.
APMIS. 2023 Aug;131(8):394-402. doi: 10.1111/apm.13329. Epub 2023 May 22.
Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.
动物和人体组织在生理和病理生理学研究中被广泛应用。由于伦理考虑和可用性低，最大限度地利用这些组织至关重要。因此，目的是开发一种新方法，允许对肾脏切片进行多重免疫荧光（IF）染色，以便多次重复使用同一组织切片。将石蜡包埋的肾脏切片放置在涂覆的盖玻片上，并进行多重 IF 染色。进行了五轮染色，每轮包括间接抗体标记、在宽场荧光显微镜上成像、使用洗脱缓冲液去除抗体，然后重新染色。在最后一轮中，用苏木精/伊红对组织进行染色。使用这种方法，可以标记肾单位中的管状段、血管和间质细胞。此外，通过将组织放在盖玻片上，使用传统的宽场荧光显微镜和 60x 油浸物镜获得类似共聚焦的分辨率。因此，使用标准试剂和设备，对石蜡包埋的组织进行多重 IF 染色，增加了 Z 分辨率。总之，该方法提供了省时的多重 IF 染色，并允许获取多个蛋白质的定量和空间表达信息，随后评估组织形态。由于这种多重 IF 方案的简单性和综合有效性，它有可能补充标准 IF 染色方案并最大限度地利用组织。
