Chemical Pretreatment of Growth Plate Cartilage Increases Immunofluorescence Sensitivity
Chemical Pretreatment of Growth Plate Cartilage Increases Immunofluorescence Sensitivity
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DOI：
10.1369/0022155411400869
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发表时间：
2011-04-01
影响因子：
3.2
通讯作者：
Dudley, Andrew T.
中科院分区：
文献类型：
--
作者：
Ahrens, Molly J.;Dudley, Andrew T.
生长板软骨中蛋白质的免疫荧光检测通常是不成功的，因为先天性自发荧光、固定剂诱导的荧光和致密的软骨基质，其可以抑制抗体渗透。为了克服这些局限性，作者测试了各种化学预处理，包括自体荧光猝灭剂硼氢化钠、煮沸柠檬酸钠的抗原修复方法、糖降解酶（透明质酸酶、肝素酶和软骨素酶）和蛋白水解酶蛋白酶XXIV。在这里，作者表明，在大多数情况下，软骨中的背景荧光是高质量成像的主要障碍。阻断样本的固有荧光与特定的预处理相结合，允许使用以前在生长板中不产生鲁棒信号的抗体进行可视化。每种抗体都需要化学预处理的特定组合，必须凭经验确定以达到最佳染色水平。本手稿包含在线补充材料，请访问http://www.jhc.org在线查看这些材料。（J Histochem Cytochem 59：408-418，2011）
Immunofluorescence detection of proteins in growth plate cartilage is often unsuccessful because of innate autofluorescence, fixative-induced fluorescence, and dense cartilage matrix, which can inhibit antibody penetration. To overcome these limitations, the authors have tested various chemical pretreatments, including the autofluorescence quencher sodium borohydride, the antigen retrieval method of boiling sodium citrate, sugar-degrading enzymes (hyaluronidase, heparinase, and chondroitinase), and the proteolytic enzyme protease XXIV. Here the authors show that, in most cases, background fluorescence in cartilage is the primary obstacle to high-quality imaging. Blocking intrinsic fluorescence of the specimen in combination with specific pretreatments allows visualization using antibodies that previously did not generate a robust signal in the growth plate. Each antibody requires a specific combination of chemical pretreatments that must be empirically determined to achieve optimal staining levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 59:408-418, 2011)