Histochem Cell Biol. 2013 Oct;140(4):423-42. doi: 10.1007/s00418-013-1133-6.
Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.
过氧化氢酶和 ABCD3 常用于定位过氧化物酶体的形态学实验。然而，它们的丰度高度依赖于代谢需求，这降低了仅基于这些蛋白质分析过氧化物酶体丰度和分布的有效性。因此，我们试图找到一种可以在各种物种、组织、细胞类型和实验设计中独立于过氧化物酶体代谢使用的蛋白质作为过氧化物体的最佳标志物。我们发现生物发生蛋白过氧化物酶体 14（PEX14）在每个过氧化物体的膜中含量相当，最适合用于免疫印迹、免疫组织化学、免疫荧光和免疫电子显微镜。使用针对 PEX14 的抗体，我们可以在各种哺乳动物组织切片（来自小鼠、猫、狒狒和人的下颌下腺和肾上腺、肾脏、睾丸、卵巢、脑和胰腺）和细胞培养物（原代细胞和细胞系）中可视化过氧化物体，其过氧化氢酶含量几乎无法检测到。过氧化物体的标记与过氧化氢酶的标记相似，组织分布与线粒体酶线粒体超氧化物歧化酶相似（两者都负责降解活性氧），而 ABCD3 仅在参与脂质代谢的细胞中表现出明显的标记。我们通过使用量子点™来提高我们的方法的灵敏度，与经典荧光染料相比，量子点™具有更高的发射产率，并且不易光漂白，从而可以更准确地定量，而不会由于个体过氧化物体的异质性而导致人为错误。我们得出结论，PEX14 确实是各种组织和细胞类型中过氧化物体标记的最佳标志物，以一致的方式进行比较形态计量学分析。
